Analiza regulacije proteinaznih gena prirodnog izolata: Lactococcus lactis subsp. lactis BGIS29
Analyses of prt genes regulation in the natural isolate Lactococcus lactis subsp. lactis BGIS29
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Prirodni izolat Lactococcus lactis subsp. lactis BGIS29 produkuje kiselu proteinazu
PI tipa i bakteriocin IS29. Prisustvo kazitona u minimalnom medijumu (MM) ima specifičan
uticaj na regulaciju aktivnosti gena za proteinazu soja BGIS29. Transkripcione genske fuzijc
sa E. coli ß-glukuronidaznim genom (gusA) su korišćene za izučavanje medijum zavisne
ekspresije promotora prt gena (VpnP i PprtM). Merenjem aktivnosti enzima ß-glukuronidaze i
kvantitativnom Northern blot analizom je pokazano da je aktivnost oba (VprtP i ?pnM)
promotora kontrolisana kazitonom na transkripcionom nivou, ali je stepen njihove regulacije
različit. Kada PpnF promotor kontroliše ekspresiju gusA gena, aktivnost enzima
ß-glukuronidaze lineamo opada sa porastom koncentracije kazitona u medijumu za rast
bakterija (0.1% do 2%). U slučaju PpnM promotora regulacija nije tako jaka kao u slučaju
PpnP promotora. Visoka koncentracija kazitona prisutna u MM ima inhibitoran efekat na
aktivnost P prtM promotora, ali ...nema lineame korelacije. Deleciona analiza regulatomog
regiona prt gena je pokazala da je celokupna informacija neophodna za punu aktivnost i
regulaciju P pnM promotora sadržana u sekvenci regiona dužine 180 bp koji sadrži oba prt
promotora.
Izucavan je uticaj nukleotidne sekvence na krivljenje DNK. Eksperimenti
pokretljivosti DNK fragmenata u gelu su korišćeni u kombinaciji sa kompjuterskom
predikcijom potencijalnih zakrivljenih regiona DNK. Kompjuterska predikcija krivljenja
DNK je pokazala da region uzvodno od -35 i -10 regiona Pprtp promotora sadrži potencijalni
centar krivljenja. Eksperimenti pokretljivosti DNK fragmenata u gelu su pokazali da je PCR
fragment veličine 350 bp, koji sadrži kompletan regulatomi region, zakrivljen. Koeficijent
retardacije (R) je 1.2 u odsustvu MgCl2 i 1.4 u prisustvu MgCl2. Kraći PCR fragment (270
bp) koji sadrži oba prt promotora je, takode, zakrivljen, R je 1.1 u odsustvu MgCl2 i 1.5 u
prisustvu MgCl2.
Inaktivacija (“knock-out”) potencijalnog regulatomog gena, prtR, je uradena
insercijom plazmida pGGiostOzISN/ u hromozom soja Lactococcus lactis NZ9000.
Merenjem aktivnosti enzima ß-glukuronidaze je pokazano da nema aktivnosti PpnP
promotora u ”knock-out” soju. Rezultati ukazuju da je potencijalni regulatomi gen aktivator.
Natural isolate Lactococcus lactis subsp. lactis BGIS29 produces the Pi-type
proteinase and bacteriocin IS29. The presence~of uasitone in chemically defined medium
(CDM) has a specific influence on the regulation of the proteinase activity in the strain
BGIS29. Transcriptional gene fusion’s with E. coli ß-glucuronidase gene (gusA) were used
to study medium dependent expression of both ¥pnP and ¥prtM promoters. The
ß-glucuronidase.assays and the quantitative Northern blot analysis showed that activities of
PpnP and VpnM promoters are controlled by casitone at the transcriptional level but their
regulation is occurring in different manner. When the ¥prtP promoter controlled the gusA
gene expression, the ß-glucuronidase activity was gradually decreasing with increase of
casitone in the growth medium (0.1% to 2%). In the case of the ¥ pnM promoter the regulation
is not that strong as in the case of the ¥ prtP promoter. Higher concentrations of casitone
present in CDM had an inhi...bitory effect on the ¥pnM promoter but there is no linear
correlation. Deletion analysis of regulatory region of the prt genes revealed that all
information needed for full activity and regulation of the ¥ prtM promoter is retained within
180-bp region that contains both the PortP and P^^promoters sequences.
The influence of nucleotide sequence context on the DNA bending was investigated.
Gel mobility experiments are used together with the computer-assisted prediction of a
putative DNA bending. Computer-assisted prediction of the DNA bending showed that the
region upstream of the —35 and -10 regions of the ¥prtP promoter contains a putative center
of the bending. Gel mobility experiments revealed that the PCR fragment of 350 bp
containing intact regulatory region is curved, the retardation value (R) was 1.2 in the
absence of the MgCl2 and 1.4 in the presence of the MgCl2. Shorter PCR fragment (270 bp)
containing both prt promoters is also curved, R were 1.1 and 1.5 in the absence and
presence of MgCl2, respectively.
A knockout of a putative regulatory gene, prtR, was done by insertion of the plasmid
pG+host9::IS.S7 into chromosome of the strain Lactococcus lactis NZ9000. There was no the
ß-glucuronidase activity controlled by ¥prtP promoter in the knock-out strain. The results
indicates that a putative regulatory gene could be an activator.