Analyses of prt genes regulation in the natural isolate Lactococcus lactis subsp. lactis BGIS29

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 inhibitory 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

Lactobacillus extra cellular proteinases

U okviru veće kolekcije prirodnih izolata mezofilnih i termofilnih laktobacila, identifikovan je određen broj izolata koji sintetišu ekstracelularne proteinaze. Sve testirane proteinaze mezofilnih laktobacila pripadaju serinskoj klasi i hidrolizuju samo beta-kazein, sa izuzetkom proteinaze soja Lactobacillus divergens BG742 koja hidrolizuje sve tri kazeinske frakcije i koja, za razliku od ostalih, najefikasnije vrši hidrolizu supstrata na baznoj pH vrednosti. Totalna DNK sojeva Lactobacillus paracasei subsp. paracasei BGLi17 i BGLi18 hibridizuje sa laktokokalnim proteinaznim probama. Restrikcione mape proteinaznih regiona ovih sojeva pokazuju visoku homologiju sa laktokokalnim proteinaznim regionima. Razdvajanje genomske DNK ovih sojeva na PFGE i hibridizacija sa laktokokalnim proteinaznim probama pokazuje da su, za razliku od laktokoka, njihovi proteinazni geni locirani na hromozomu. Termofilni soj Lactobacillus delbruckli subsp. bulgaricus BGPF1 sintetiše proteinazu koja takođe hidrolizuje samo beta-kazein i koja, sudeći na osnovu rezultata PCR analize, pripada tipu koji je kod drugih sojeva ove vrste ranije okarakterisan. Soj Lactobacillus acidophilus BGRA43 sintetiše proteinazu koja hidrolizuje sve tri kazeinske frakcije, ali za koju se na osnovu hibridizacije i PCR analize može reći da ne pripada nijednom do sada opisanom tipu. .Within the large collection of natural isolates of mesophilic and thermophilic lactobacilli, some of the isollates were identified as extracelular proteinase producers. All tested proteinases from mesophilic lactobacilli belong to the serine class and hydrolize beta-casein only, except the proteinase from the strain Lactobacillus divergens BG742 which hydrolyze all three casein fractions, and which is, in contrast to the others, more efficient at basic pH values. Total DNA from strains Lactobacillus paracasei subsp. paracasei BGLi17 and BGLi18 hybridizes with the probes from lactococcal proteinase gene regions. On the basis of PFGE analysis of genomic DNA of these strains and hybridization with lactococcal proteinase probes, it could be suggested that their proteinase gene regions are chromosomaly located. Thermophilic strain Lactobacillus delbrueckii subsp. bulgaricus BGPF1 produces the proteinase which also hydrolyzes only beta-casein, and which is, according to the PCR analysis, of the same type already described in other strains of this species. Proteinase from strain Lactobacillus acidophilus BGRA43 hydrolyzes all three casein fractions, and on the basis of hydrodization and PCR analysis this enzyme is different from the proteinases described so far.