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Studies in the physiology of the genus BacillusCoffee, William B. January 1942 (has links)
Twenty-eight different species of the genus Bacillus have been examined both as to morphological characteristics and physiological reactions. The physiological reactions tested include gelatin liquefactions, acetyl-methyl-carbinol production, nitrate reduction and 36 different carbohydrate fermentations.
The action of all 78 strains of the 28 species was also determined on peptone and graphs have been prepared comparing this action with that of the organisms of the carbohydrates.
Definite explanations are given for many of the inconsistencies existing in the literature for the fermentation reactions of the bacilli.
The results obtained are compared with those recorded by three previous workers, namely Fitzgerald (8), Soriano (29) and Bergey’s Manual (1). Reasons have been advanced to explain cases of disagreement. These are concerned mainly with the indicator used and the failure to take into account the action of the organisms under investigation upon the basal medium.
Where it was felt that a representative number of strains were examined, definite conclusions as to the ability of the organisms to dissimilate various carbohydrates are drawn.
A key for the classification of the B. mesentericus group has been formulated based entirely on carbohydrate fermentations. The procedure followed appears to be applicable to other groups as well as to the entire genus. / Master of Science
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Sporulation and toxin formation in Bacillus sphaericus 1593Madhekar, Nandu M. January 1983 (has links)
The effects of temperature, dissolved oxygen, and pH on the growth, sporulation, and mosquito larval toxin formation by B. sphaericus 1593, were investigated in shaken flasks and a fermenter. The bacteria grew well at temperatures of 25 to 40°C, however, toxin-formation and sporulation were poor at temperatures above 30°C. Cell lysis became evident after about 30 hours into the fermentation, particularly in case of high temperatures. Mature spores, as well as most of the toxin, were formed by 24 to 30 hours of growth.
Controlling the pH at 7.0, gives about ten-fold more toxicity as compared to experiments with no pH control. Simultaneous assay of ammonia concentration indicated a close parallel between the ammonia and pH profiles.
Assays of carbohydrate, protein, and phosphorous indicated that none of these nutrients were limiting at any time, thus none of them could have been the limiting nutrient that triggers sporulation.
It was found that dissolved oxygen concentrations dropped nearly to zero in case of fermentation with sparged air, but were significant in the case of fermentation with sparged oxygen even at their minimum. The toxicity was approximately equal in both the cases, however sporulation was poor in the case of experiments sparged with pure oxygen. Measurements of oxygen uptake rates confirmed that the cells respire more when the cells are in the vegetative phase of growth. / M.S.
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Detection of NheA from Bacillus spp. in food and soil isolates using real-time and rep-PCR / Detection of non-hemolytic enterotoxin A from Bacillus spp. in food and soil isolates using real-time and rep-polymerase chain reactionBeer, Matthew R. 06 August 2011 (has links)
Bacillus cereus is traditionally thought to be the only member of its genus accepted as a pathogen in foods like grains, fruits, vegetables and milk due to the presence of the nonhemolytic (Nhe) operon. However, many other Bacillus spp. may also harbor the Nhe operon and be pathogenic. Real-time PCR targeted the nheA gene in 37 samples obtained from food, soil, and reference cultures by analyzing the standard deviations of melt peaks. Rep-PCR was used to compare the banding patterns of each sample against B. cereus ATCC14579 and three B. thuringiensis strains to “fingerprint” each isolate. Of the original 43 isolated tested, 37 were Gram-positive rods. The remaining six samples were Gram-positive cocci. Twenty-five of the 37 Gram-positive Bacillus spp. were nheA positive, while twelve were negative. Many of the nheA positive strains were species not previously known to contain Nhe, and were capable of causing gastroenteritis in consumers. / Department of Biology
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Cloning of a novel esterase gene from Bacillus pumilus and its characterisation in Escherichia coliPieterse, Anton 03 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Esterases play a variety of roles in nearly every aspect of life ranging from cellular
metabolism, signal transduction to defence mechanisms in plants. One aspect of
esterases that recently is receiving more attention is the role esterases play in the
.degradation of plant material. With fossil fuels (coal and oil) estimated to run out in
the next 20 to 30 years, renewable sources such as plant biomass are becoming
increasingly important. Plant biomass contains hemicellulosic and cellulosic
materials that need to be degraded to their different constituents before they can be
optimally used for the production of commodities.
Although the enzymes needed to hydrolyse the xylan backbone (xylanases and
P-xylosidases) are important, enzymes that remove side chains from the polymer are
equally important. They facilitate hydrolysis by xylanases and P-xylosidases and will
improve the availability of monomeric sugars for utilisation when used in conjunction
with other xylanolytic enzymes. Many of these side-chains are esters and they need
to be removed through the action of esterases, either directly from the xylan backbone
or from shorter xylo-oligomers.
An existing genomic DNA library of Bacil/us pumilus in Escherichia coli was
screened for the presence of an acetyl esterase encoding gene. Positive clones were
identified by the formation of clearing zones on plates containing glucose
pentaacetate. Plasmid DNA was isolated from a positive E. coli clone. The DNA
insert was sequenced and found to contain two open reading frames, one of which
encoded a novel esterase (estA). Using different primers the gene was amplified by polymerase chain reaction and inserted into an inducible expression vector (pKK223-
3) for expression in E. coli. The plasmid was introduced into E coli and the esterase
activity determined, using the chromogenic substrate a-naphthyl acetate. Activity
levels decreased shortly after induction with IPTG and therefore plasmid pAP4 was
used for enzymatic assays. Cultures containing plasmid pAP4 produced extracellular
activity of 2.5 nkatal/ml. The pH and temperature optima as well as temperature
stability of the enzyme was determined. The enzyme exhibited optimal activity at pH
6 and 60°C and was stable at 60°C after 2 h. Enzyme assays on different substrates
yielded activity on methylumbelliferyl butyrate and methylumbelliferyl acetate in
addition to the glucose pentaacetate and a-naphthyl acetate. The estA gene was
cloned into a yeast expression vector between the PGK promoter and terminator
sequences for expression of the gene in Saccharomyces cerevisiae. The estA open
reading frame was also fused to the MFa 1 secretion signal for secretion of the protein
from S. cerevisiae. The expression vector was successfully transformed into S.
cerevisiae, but no extracellular activity was detected. Only low intracellular activity
of 0.260 nkatal/ml was detected in S. cerevisiae. / AFRIKAANSE OPSOMMING: Esterases speel 'n verskeidenheid van rolle in feitlik elke aspek van lewe, van sel
metabolisme, sein transduksie tot verdedigingsmeganismes in plante. Een aspek van
esterases wat al hoe meer aandag geniet, is die rol wat esterases in die afbraak van
plant en plantmateriaal speel. Met olie- en steenkoolbronne wat na beraming oor 20
tot 30 jaar tot niet sal gaan, raak die rol wat hernubare bronne speel al hoe
belangriker. Plantbiomassa bevat sellulose en hemisellulose wat tot die verskillende
komponente afgebreek moet word voordat dit optimaal vir die vervaardiging van
produkte aangewend kan word.
Alhoewel die ensieme wat vir die hidrolise van die xilaanruggraat benodig word,
(xilanases en ~-xulosidases) belangrik is, is die ensieme wat die sygroepe vanaf die
polimeer verwyder ewe belangrik aangesien hulle die hidrolise deur xilanases en
~-xulosidases bevorder. Wanneer hulle saam met die ander xilanolitiese ensieme
gebruik word, sal hulle die beskikbaarheid van monomeriese suikers vir fermentasie
verhoog. Baie van hierdie sygroepe is esters en hulle word deur die aksie van
esterases verwyder, of direk van die ruggraat, ofvanafkorter xilo-oligosakkariede.
'n Bestaande genoom DNA biblioteek van Bacillus pumilus in Escherichia coli is vir
die teenwoordigheid van 'n asetielesterase-koderende geen gesif. Positiewe klone is
deur die vorming van 'n sone op plate wat glukose pentaasetaat bevat, geïdentifiseer.
Die DNA-invoeging van die positiewe E. coli-kloon se DNA-volgorde is bepaal en
twee oopleesrame is gevind waarvan een vir 'n unieke esterase (estA) kodeer. Met
behulp van verskillende inleiers is die geen met die polimerasekettingreaksie (PKR) geamplifiseer en in 'n induseerbare promotor vir uitdrukking in E. coli gekloneer. Die
plasmied is getransformeer in E. coli en aktiwiteit is bepaal deur cc-naftielasetaatte
gebruik. Vlakke van aktiwiteit het kort na induksie met IPTG weer gedaal en daarom
was plasmied pAP4 vir ensiematiese toetse gebruik. E. coli-transformante met
plasmied pAP4 het ekstrasellulêre aktiwiteit van 2.5 nkatal/ml gelewer. Die pH en
temperatuur optima sowel as die temperatuurstabiliteit van die ensiem was bepaal.
Die ensiem toon optimale aktiwiteit by pH 6 en 'n temperatuur van 60°C.
Aktiwiteitstoetse op verskillende substrate het aktiwiteit op metielumbelliferielasetaat
en metielumbelliferielbutiraat bo-en-behalwe die glukosepentaasetaat en
c-naftielasetaar getoon. Die estA geen is in uitdrukkingskasette bevattende die PGKpromotor
en-termineerder vir uitdrukking in Saccharomyces cerevisiae gekloneer.
Dit is ook agter die MFal-sekresiesein gekoppel vir sekresie vanuit S. cerevisiae.
Geen ekstrasellulêre aktiwiteit is gevind nie. Slegs intrasellulêre aktiwiteit van 0.26
nanokatal per mililiter was bepaal.
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Molecular characterization of Sulfobacillus and related organismsSchutte, Mart-Alet (Martha Aletta) 03 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Thirteen Sulfobacillus strains from different geographical locations and two
Alicyclobacillus strains were included in this study. These organisms proved to be
moderately thermophillic (two different sets of optimal temperatures of 45°C and 55°C
were found), Gram-positive, endospore forming bacteria. The pH optima of the strains
tested was pH 2.5 and the pH range lay between pH 1.5 and pH 5.0. It was established
that some strains of Sulfobacillus had the capacity for anaerobic growth when using ferric
iron as an electron donor. It was determined that S. thermosuljidooxidans was the species
found within South African biooxidation plants. Plasm ids were identified within strain
611 (S. thermosuljidooxidans) isolated from a Billiton commercial plant.
The sample of Sulfobacillus strains used in this study could clearly be divided into two
groups based on the analysis of their 16S rRNA gene sequences as well as the number of
ribosomal (rm) operons present as determined by Southern hybridization.
A system for the convenient identification of Sulfobacillus species was developed using
several of the techniques employed in this study. Preliminary identifications can be
obtained by restriction enzyme digestion of the PCR amplified 16S rRNA gene.
Confirmation of this placement can be done by comparison of the 16S - 23S rRNA
spacer region amplification band sizes. Once the preliminary identification has been
completed it is possible to place the isolate in the correct species by making use of the
differences in sugar utilization that the species exhibit. The more laborious method of
16S rRNA sequence comparisons can be undertaken if there is still any uncertainty as to
which species an isolate belongs to. Phylogenetic results obtained from the 16S rRNA gene sequence indicates that the genus
Sulfobacillus should probably be divided into two individual genera. Further information
gathered from the phylogenetic comparisons indicates that strain Riv-14 previously
assigned to S. ambivalens is more closely related to S. montseratensis. Data obtained
from 16S - 23S rRNA spacer region analysis confirms this result.
Future work includes the use of DNA-DNA hybridization studies and mol% G+C ratio's
in order verify the presence of two distinct genera as well as placing Riv-14 within the
correct species. / AFRIKAANSE OPSOMMING: Dertien isolate van die genus Sulfobacillus afkomstig van geografies verskillende areas
en twee isolate van die genus Alicyclobacillus is in die studie ingesluit. Hierdie
organismes het gewys dat hulle gematigde termofiele (twee verskillende groepe met
optimale temperature van 45°C en 50°C elk was waargeneem), Gram-positiewe,
endospoorvorrnende organismes is. Die pH optima van die isolate was pH 2.5 en die
reeks van pH waar groei moontlik was het tussen pH l.5 en pH 5.0 gelê. Dit was bewys
dat sekere van die Sulfobacillus isolate oor die vermoë beskik het om anaerobies te
respireer deur ferri yster (Fe3+) as elektron akseptor te gebuik. Dit was bepaal dat S.
thermosulfidooxidans die spesies is wat teenwoordig was in die bio-oksidasie reaktors in
Suid Afrika. Plasmiede vanuit die isolaat 611 (s. thermosulfidooxidans) afkomstig vanuit
'n Billiton komersieële reaktor, is geidentifiseer.
Die toetsmonster van Sulfobacillus isolate gebruik in hierdie studie het duidelik daarop
gewys dat daar twee groepe binne Sulfobacillus is. Hierdie stelling is gebaseer op data
afkomstig van die analiese van die 16S rRNA volgorde asook die aantal ribosomale (rm)
kopieë teenwoordig soos bepaal deur Southern klad eksperimente.
'n Sisteem vir die maklike identifikasie van Sulfobacillus spesies is ontwerp deur van
verskeie tegnieke, soos in hierdie studie toegepas, gebruik te maak. Aanvanklike
identifikasie kan verkry word deur gebruik te maak van restriksie ensiem vertering van
PKR geamplifiseerde 16S rRNA geen. Hierdie plasing van die isolaat kan bevestig word
deur die grootte van die 16S - 23S rRNA intergeniese amplifikasie produkte te vergelyk.
Sodra die aanvanklike plasing van die isolaat voltrek is, kan daar van die verskille in die
vermoëns van die spesies om sekere suikers the benut, gebruik gemaak word om die
isolaat binne die regte spesies te plaas. Die meer werksintensiewe metode van 16S rRNA
volgorde vergelyking kan gebruik word indien daar enige onsekerheid is oor by watter
spesies die isolaat hoort. Filogenetiese resultate verkry van die vergelyking van die 16S rRNA geen volgorde dui
daarop aan dat die genus Sulfobacillus waarskynlik uit meer as een genus bestaan. Die
filogenetiese data dui verder daarop dat die isolaat Riv-14 wat as 'n S. ambivalens
geklassifiseer is, nader verwant is aan die spesies S. montseratensis. Data verkry vanaf
die 16S - 23S intergeniese gebied analiese bevestig hierdie resultaat.
Toekomstige werk sluit DNA-DNA hibridisasie en mol% Gte ratio eksperimente in om
sodoende die teenwoordigheid van meer as een genus sowel as die plasing van Riv-14 in
die korrekte spesies te bevestig.
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Molecular genetics of arsenic resistance of the biomining bacterium Acidithiobacillus ferrooxidansButcher, Bronwyn Gwyneth 12 1900 (has links)
Dissertation (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The acidophilic, chemolithoautotrophic bacterium, Acidothiobaci/lus ferrooxidans is one
of a consortium of bacteria involved in biornining, including the recovery of gold from
arsenopyrite ores. The genes conferring arsenic resistance to At. ferrooxidans were
cloned and sequenced and shown to be chromosomally located. Homologues to the arsB
(membrane located arsenite efflux pump), arsC (arsenate reductase) and arsH (unknown
function) genes from known arsenic resistance (ars) operons were identified. A fourth
gene was found to have weak homology to the ArsR-family of regulators. The arsenic
resistance genes of At. ferrooxidans are arranged in an unusual manner, with the arsRC
and arsBH genes divergently transcribed. This divergent arrangement was found to be
conserved in all four of the At. ferrooxidans strains we tested.
All of the At. ferrooxidans ars genes were expressed in Escherichia coli and the arsB and
arsC genes conferred arsenite (and antimonite) and arsenate resistance, respectively, to an
E. coli ars mutant (AW311 0). Analysis of the putative amino acid sequences of these ars
genes revealed that the ArsB from At. ferrooxidans is closely related to the ArsB proteins
from other Gram-negative bacteria. However, the ArsC protein is more closely related to
the ArsC proteins from Gram-positive bacteria. Furthermore, a functional thioredoxin
(trxA) gene was required for ArsC-mediated arsenate resistance in E. coli. This suggests
that reduction of arsenate by At. ferrooxidans has a similar reaction mechanism as that by
Gram-positive ArsC proteins. While arsH was expressed in an E. coli-derived in vitro
transcription-translation system, the presence of this gene was not required for, nor
enhanced, arsenite or arsenate resistance in E. coli. We predict that the function provided
by this gene is not required in E. coli.
While the putative ArsR from At. ferrooxidans does contain a potential DNA-binding
helix-turn-helix (HTH) domain, it does not contain the arsenite binding motif
(ELCVCDL), required for response to the presence of inducer. Instead, the ArsR-like
protein from At. ferrooxidans is related to a group of unstudied ArsR-like proteins that
have been associated with other ars-like genes identified during genome sequencing
projects. Using arsB-lacZ, arsC-lacZ, and arsR-lacZ fusions, it has been shown that this atypical ArsR protein from At. ferrooxidans did repress expression from the arsBH and
arsRC promoters and that this repression was relieved by the presence of either arsenite or
arsenate. Deletion of 19 amino acids from the C-terminus of the ArsR protein did not
affect regulation, while deletion of a further 28 amino acids inactivated ArsR. Northern
blot hybridization confirmed that expression of the arsRC and arsBH transcripts is
increased in the presence of either arsenite or arsenate.
This study is the first to show that the ars genes from the acidophilic biorning bacterium
At. ferrooxidans are able to be studied in the neutrophilic bacterium, E. coli. We have
also shown that the atypical ArsR found in this ars operon is able to regulate expression
of these genes in response to arsenic, despite not containing the arsenite binding domain,
suggesting that this protein senses arsenic by a different mechanism to that used by the
ArsR family members already studied. / AFRIKAANSE OPSOMMING: Acidothiobacillus ferrooxidans, 'n asidofiliese, chemolitotrofiese bakterium, is een van 'n
konsortium bakterieë betrokke by biologiese ontgunnig ("biomining") asook by die
herwinning van goud uit arsenopiriet erts. Die gene wat aan At. ferrooxidans
weerstandbiedendheid teen arseen verleen, is gekloneer. Die DNA-volgorde van hierdie
gene is bepaal en daar is bewys dat die gene op die chromosoom geleë is. Homoloë van
die arsB (membraan geleë pomp wat arseniet uitpomp), arsC (arsenaat reduktase) en die
arsH (funksie onbekend) gene is in bekende arseenweerstanbiedheidsoperons (arsoperons)
geïdentifiseer. Verder is daar 'n vierde geen geïdentifiseer wat lae homologie
met die ArsR-familie van reguleerders toon. At. ferrooxidans se ars gene is op 'n
ongewone manier gerangskik met twee van die gene, arsRC en arsBH wat lil
teenoorgestelde rigtings getranskribeer word. Hierdie rangskikking van gene IS
waargeneem in al vier die At. ferrooxidans rasse wat getoets is.
Al die At. ferrooxidans ars gene is in Escherichia coli uitgedruk. Die arsB en arsC gene
het aan 'n E. coli ars mutant (AW311 0) weerstandbiedendheid teen aseniet, antimoniet en
arseen verleen. Analiese van die afgeleide aminosuurvolgorde van die ars proteïene het
getoon dat die At. ferrooxidans ArsB naby verwant aan die ArsB-proteïene van ander
Gram negatiewe bakterieë is. In teenstelling hiermee, is gevind dat die ArsC-proteïene
nader verwant aan die ArsC-proteïene van Gram positiewe bakterieë is. Daar is ook
gevind dat 'n funksionele tioredoksien (trxA) geen vir ArsC-bemiddelde arsenaat
weerstandbiedendheid in E.coli benodig word. Dit dui daarop dat die meganisme van
arsenaatreduksie deur At. ferrooxidans soortgelyk is aan die ArsC-proteïen-meganisme
van Gram positiewe bakteriee. In vitro studies met behulp van 'n E. coli gebaseerde
transkripsie-translasie sisteem het getoon dat arsH nie nodig is vir arsenaat of aseniet
weerstanbiedendheid in sensitiewe E.coli rasse nie en ook nie help om weerstand in
hierdie rasse te verhoog nie. Daarom kan daar aangeneem word dat die funskie van die
arsH geen nie deur E. coli benodig word nie.
Die vermeende ArsR van At. ferrooxidans bevat 'n potensiële DNA-binding heliks-draaiheliks
motief, maar nie die arsiniet binding motief (ELCVCDL) wat nodig is vir reaksie in
die teenwoordigheid van 'n induseerder nie. Die ArsA-proteïen van At. ferrooxidans is
soortgelyk aan 'n groep ArsA-proteïene wat tydens genoom DNA- volgordebepalingsprojekte geïdentifiseer is. Hierdie groep gene is egter nog nie verder
bestudeer nie. Deur gebruik te maak van 'n stel fusie gene, arsB-IacZ, arsC-IacZ en arsRlacZ
kon daar bewys word dat die ongewone ArsH-proteïen van At. ferrooxidans
uitdrukking van arsBH en arsRC onderdruk en dat die onderdrukking deur arseniet of
arsenaat opgehef kan word. Delesie van die eerste 19 aminosure vanaf die C-terminus
van die ArsA-proteïen het geen uitwerking op die regulering van die proteïen nie, maar
delesie van 'n vedere 28 aminosure het ArsR geïnaktiveer. Verhoogde vlakke van
transkripsie van arsRC en arsBH in die teenwoordigheid van arseniet en arsenaat is met
behulp van Noordelike kladanalise bewys.
Hierdie is die eerste studie waarin daar bewys word dat die ars gene van die asidofiliese
bakterium Atferrooxidans in die neutrofiliese bacterium E. coli bestudeer kan word. Daar
is ook bewys dat ten spyte daarvan dat die ArsR in die ars operon nie 'n arseniet
bindingsdomein het nie, dit die uitdrukking van die gene in hierdie operon reguleer in
reaksie op arseen. Dit dui dus daarop dat hierdie proteïen op arseen in die omgewing
reageer met behulp van 'n meganisme wat verskil van die ArsR-proteïene wat tot dusver
bestudeer is.
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Selección de Pseudomonas sp., Bacillus sp. y actinomicetos productores de Ácido Indol Acético (AIA) aislados de “Biol” de elaboración artesanal provenientes de Lima y HuancayoLino Villanueva, Gladys Liliana January 2011 (has links)
El “biol” es un abono líquido rico en fitohormonas, como las auxinas o mejor conocido
como Ácido Indol Acético (AIA). Éste ácido estimula el desarrollo de las plantas y en
general, la germinación de semillas. El uso del biol, en la agricultura peruana ha
cobrado mucha importancia, pues asegura mayor rendimiento de la producción,
incrementando a la vez la calidad de los cultivos y sobretodo ofreciendo alimentos
libres de productos químicos. Para esta investigación se prepararon 22 muestras de biol
artesanal con composiciones variables instalados en Lima y Huancayo. Se realizaron
10 muestreos durante un periodo de 150 días de biodigestión para seleccionar
Pseudomonas sp., Bacillus sp. y actinomicetos productores de AIA, los cuales fueron
identificados por sus características culturales y pruebas preliminares. Posteriormente
con todas las cepas seleccionadas se realizó la prueba cualitativa y cuantitativa del
AIA. El 20% de Pseudomonas sp., 17,07% de Bacillus sp. y 21,35% de actinomicetos
aislados resultaron positivos a la prueba de AIA y fue posible cuantificar su producción.
Asimismo, se realizó una prueba de efectividad del AIA producido por nueve bacterias
para evaluar su efecto promotor de crecimiento sobre el hipocotilo y la radícula en
semillas de Lactuca sativa L. (lechuga). El análisis estadístico de las pruebas señaló
que los resultados son altamente significativos, mostrando los mejores resultados la
cepa PSIV-1-L para el hipocótilo y la cepa PSII-7-L para la radícula. Se comprobó así
la presencia de bacterias productoras de AIA en las muestras de biol de preparación
artesanal y a la vez fue posible evidenciar un efecto positivo sobre la germinación in
vitro de semillas de Lactuca sativa L. (lechuga). / Tesis
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Regulating polysaccharide synthesis in bacteriaChen, Donald D. 02 September 1993 (has links)
Graduation date: 1994
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Characterization of selected Bacillus isolates exhibiting broad spectrum antifungal activity.Tewelde, Teklehaimanot Weldeslasie. January 2004 (has links)
The genus Bacillus is comprised of Gram-positive, rod-shaped, spore-forming bacteria which are
well known for their ability to produce a diverse array of antimicrobial compounds. Ofparticular
interest is the ability of certain strains to produce antifungal compounds. Such organisms have the
potential for application in agriculture where they can be used as biocontrol agents against
selected plant pathogenic fungi. A study was undertaken to further characterize selected Bacillus
isolates that exhibit broad spectrum antifungal activity.
Dual culture bioassays were used to screen seven selected Bacillus isolates for activity against
four plant pathogenic fungi in vitro. All isolates were able to inhibit the pathogens to varying
degrees. Two isolates, R29 and B81, were selected for further testing and characterization.
Further bioassays were performed on five complex nutrient media which were adjusted to pH S.S
and 7, and both incubated at 2SoC and 30°C" respectively. It was found that pH and media
composition showed significant influences on the antifungal activities of the isolates tested, but
that a SoC temperature difference in incubation temperature did not. Tryptone soy agar was found
to give rise to the largest inhibition zones.
Both isolates were tentatively identified using standard biochemical and morphological tests.
Based on its phenotypic characteristics, R29 was identified as a strain of B. subtilis. B81 proved
to be more difficult to assign to a specific group or species of Bacillus, though B. subtilis and
B. licheniformis were considered to be the nearest candidates. Genomic DNA was extracted from
both isolates and a portion of each of their 16s rDNA genes were amplified and sequenced for
homology testing against the GeneBank database. Homology testing confirmed that both isolates
were members of the genus Bacillus and most probably strains of B. subtilis. The DNA fragment
used for sequencing proved to be too small to give conclusive identification of the isolates.
Isolate R29 was selected for further characterization of its antifungal compound/so Growth curve
studies using a defined synthetic medium showed that antifungal activity arose during the
stationary phase and appeared to be closely linked to sporulation. The antifungal component of
cell free culture supematant was extracted using various methods including thin layer
chromatography, acid precipitation, hydrophobic interaction chromatography and methanol
extractions. High performance liquid chromatography (HPLC) analysis of extracts from acid precipitation and hydrophobic interaction chromatography revealed two active peaks indicating
that at least two antifungal compounds were produced. Methanol extracted samples produced the
cleanest sample extract but only revealed one active peak from the HPLC fraction .
Nuclear magnetic resonance analysis of purified samples indicated that the antifungal
compound/s have aromatic complex and peptide structures. The extracted antifungal compounds
were Protease K resistant and found to be thermostable at temperatures ranging 80-121oC, and,
were active at pH ranges of 3-13. The antifungal compounds were found to exhibit similar
properties to known antifungallipopeptides i.e. iturin A and fengycin A and B.
Further characterization and identification of the active compounds is recommended usmg
methods such as liquid chromatography mass spectrometer and matrix-assisted laser desorption
ionisation time-of- flight.
The results presented in this dissertation provide a basis from which antifungal compounds
produced by strains ofBacillus can be further characterized. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.
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Development of a Rep-PCR screening assay for enterotoxigenic Bacillus spp. in naturally contaminated food / Development of a repetitive element palindrome-polymerase chain reaction screening assay for enterotoxigenic Bacillus spp. in naturally contaminated foodCooper, Robin M. January 2004 (has links)
Several powdered food products were screened using repetitive element palindrome PCR (rep-PCR) for the presence of enterotoxin producing species of Bacillus. Samples from these products were screened by being placed into a tryptone-peptoneglucose-yeast enrichment medium (TPGY), heat-treated, and shake-incubated. DNA was extracted using a modification of established protocol, leading to the development of an optimized method for each food system. Purified DNA was amplified through rep-PCR using extragenic sequence-targeting primers and optimized for each food product. Amplified PCR products were analyzed electrophoretically and viewed using an ultraviolet photodocumentation system. Bacillus cereus positive control DNA fingerprints were compared to banding patterns from enriched food samples, revealing the presence of the typical diagnostic 1,230 bp band in non-fat dry milk (NFDM). Restriction Fragment Length Polymorphism (RFLP) with Alu I restriction enzyme was performed on the 1230 bp diagnostic band from NFDM and displayed a profile consistent with Bacillus cereus positive control. RPLA (Reverse Passive Latex Agglutination) and BDE ELISA (Bacillus Diarrhoeal Enterotoxin Enzyme Linked Immunosorbent Assay - Tecra Diagnostics) confirmed the presence of HBL and NHE enterotoxin production in NFDM, Coffee creamer, infant milk formula, and two lecithin samples. / Department of Biology
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