Biomedical subjects

L Topisirovic

Publications and source records attributed to L Topisirovic.

At least 19 recordsLinked to original sources

PubMed2001

Casitone-mediated expression of the prtP and prtM genes in Lactococcus lactis subsp. lactis BGIS29.

Casitone added to chemically defined medium (CDM) specifically influenced the regulation of the proteinase activity in the natural isolate Lactococcus lactis subsp. lactis BGIS29. Comparative analysis of the influence of casitone present in CDM on the proteolytic activity of strain BGIS29 and of L. lactis subsp. cremoris strains SK11 and Wg2 indicated that the proteolytic activity of strains BGIS29 and SK11 is casitone-dependent, whereas that of strain Wg2 is not. The regulatory region of the prt genes of strain BGIS29 was cloned and sequenced. The nucleotide sequence of the prt regulatory region of strain BGIS29 was distinctly different from that of L. lactis subsp. cremoris strains SK11 and Wg2. Transcriptional gene fusions with the Escherichia coli beta-glucuronidase gene ( gusA) were used to study medium-dependent expression of two divergent prtP and prtM promoters of strain BGIS29 (designated P (prtP) and P (prtM), respectively). beta-Glucuronidase assays and Northern blot analysis showed that the activities of P (prtP) and P (prtM) are controlled by casitone at the transcriptional level.

Amino Acid Sequence↗

PubMed2001

Characterization of cell envelope-associated proteinases of thermophilic lactobacilli.

The proteolytic activities of two natural isolates of thermophilic lactobacilli, Lactobacillus acidophilus BGRA43 and Lact. delbrueckii BGPF1, and Lact. acidophilus CH2 (Chr. Hansen's strain) and Lact. acidophilus V74 (Visby's strain), were compared. Results revealed that optimal pH for all four proteinases is 6.5, whereas temperature optimum varied among proteinases. Determination of caseinolytic activity done under optimal conditions for each strain revealed that the CH2 and V74 proteinases completely hydrolysed both alphaS1-casein and beta-casein, showing very low activity towards kappa-casein. The BGPF1 proteinase completely hydrolysed only beta-casein. The BGRA43 proteinase completely hydrolysed all three casein fractions. The proteolytic activities of whole cells were inhibited by serine proteinase inhibitors, suggesting that all four strains produce serine proteinases. DNA-DNA hybridization and PCR analysis showed that BGPF1 contains the prtB-like proteinase gene. Characterized thermophilic strains BGPF1 and BGRA43 were successfully used as starter cultures for production of yoghurt and acidophilus milk, respectively.

Bacterial Proteins↗

PubMed2001

Surface expression of the conserved C repeat region of streptococcal M6 protein within the Pip bacteriophage receptor of Lactococcus lactis.

The C repeat region of the M6 protein (M6c) from Streptococcus pyogenes was expressed within the Pip bacteriophage receptor on the surface of Lactococcus lactis. M6c was also detected in the culture medium. The pip-emm6c allele was integrated into the chromosome and stably expressed without antibiotic selection. The level of cell-associated surface expression of PipM6c was 0.015% of total cellular protein. The amount of PipM6c on the cell surface was increased about 17-fold by expressing pip-emm6c from a high-copy-number plasmid. Replacing the native pip promoter with stronger promoters isolated previously from Lactobacillus acidophilus increased surface expression of PipM6c from the high-copy-number plasmid up to 27-fold. Concomitantly, the amount of PipM6c in the medium increased 113-fold. The amount of PipM6c did not vary greatly between exponential- and stationary-phase cultures. Western blots indicated that the full-length PipM6c protein and most of the numerous proteolytic products were found only on the cell surface, whereas only one proteolytic fragment was found in the culture medium.

Antigens, Bacterial↗

PubMed2000

Molecular analysis of mutated Lactobacillus acidophilus promoter-like sequence P15.

The promoter-like sequence P15 that was previously cloned from the chromosome of Lactobacillus acidophilus ATCC 4356 is active in Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus acidophilus, and Escherichia coli, but not in Lactococcus lactis. N-methyl-N-nitroso-N-guanidine (MNNG) mutagenesis of P15 was used to select for a promoter active in L. lactis MG1363. Molecular analysis of the mutated promoter (designated P16) revealed a 90 bp deletion and a T-->A transversion. This deletion, in combination with the addition to the transversion, created a promoter with putative -35 and -10 hexamers identical to the consensus promoter sequence found in E. coli and Bacillus subtilis vegetative promoters. The activity of P16 was measured by its ability to promote chloramphenicol resistance in different bacteria when inserted in the promoter-probe plasmid pBV5030 (designated pLA16). The MIC of chloramphenicol in L. lactis, L. reuteri, L. plantarum, E. coli, and L. acidophilus harbouring pLA16 were 30, 170, 180, > 500, and 3 micrograms/mL, respectively. This represents an increase in promoter activity compared to P15 in L. reuteri of 3-fold, in L. plantarum of 9-fold, and in E. coli of at least 2.5-fold, but a decrease in L. acidophilus of 7-fold.

Anti-Bacterial Agents↗

PubMed1998

Characterization of natural isolate Lactobacillus acidophilus BGRA43 useful for acidophilus milk production.

Lactobacillus acidophilus BGRA43 was selected from a set of human origin isolates of Lact. acidophilus strains for the highest growth rates and antagonistic effect against both Gram-positive and Gram-negative bacteria. The strain BGRA43 also exhibited an inhibitory effect on the growth of Clostridium sporogenes. Inhibition of this strain seems to be due to lactic acid production rather than hydrogen peroxide or bacteriocin. Growth of Lact. acidophilus BGRA43 in non-fat skim milk for 6 h at 37 degrees C resulted in a lowering of the pH value to 4.53. Besides the fast acidification, this strain generated a high viscosity of skim milk. These characteristics make the strain BGRA43 attractive for acidophilus milk production. Lactobacillus acidophilus BGRA43 produces an extracellular proteinase. Whole cells efficiently degraded casein for 3 h at 37 degrees C especially alpha- and beta-casein fractions. Total DNA isolated from the strain BGRA43 did not show any hybridization with lactococcal proteinase probes indicating that this strain produces a distinctive proteinase.

Animals↗

PubMed1997

Cloning and molecular analysis of promoter-like sequences isolated from the chromosomal DNA of Lactobacillus acidophilus ATCC 4356.

Promoter-like sequences from the chromosomal DNA of thermophilic strain Lactobacillus acidophilus ATCC 4356 were cloned. Analysis of the three DNA fragments showing promoter activity, designated P3, P6, and P15, were performed in Lactobacillus reuteri, Lactococcus lactis, and E. coli. The reporter cat-86 gene was expressed in all three bacterial species under control of the fragments P3 and P6. Fragment P15 showed promoter activity only in Lactobacillus reuteri and E. coli but not in Lactococcus lactis. The three host-specific transcriptional start points (TSPs) were used when transcription of the cat-86 gene was controlled by fragment P3 in Lactobacillus reuteri, E. coli, and Lactococcus lactis. Similarly, fragment P15 initiated transcription of the cat-86 gene at two distinctive sites in Lactobacillus reuteri and E. coli. Only within fragment P6, a common TSP was used in Lactobacillus reuteri and E. coli, but different from that used in Lactococcus lactis. Each TSP was preceded by the putative -35 and -10 hexamers. Computer analysis of the fragment P3 sequence revealed the existence of divergent promoter-like sequence (P3rev) located on the complementary DNA strand. Fragments P6 and P15 were also functional in Lactobacillus acidophilus ATCC 4356 from which chromosomal DNA they were originally cloned.

Amino Acid Sequence↗

PubMed1996

Translational autoregulation of the sgm gene from Micromonospora zionensis.

The sisomicin-gentamicin resistance methylase gene (sgm) from Micromonospora zionensis (the producer of antibiotic G-52 [6-N-methyl-sisomicin]) encodes an enzyme that modifies 16S rRNA and thereby confers resistance to 4,6-disubstituted deoxystreptamine aminoglycosides. Here, we report that this gene is regulated on the translational level. The Escherichia coli lacZ gene and operon fusion system was used, and it was shown that an extra copy of the sgm gene decreases the activity of the fusion protein. These results suggested that expression of the sgm gene is regulated by the translational autorepression because of binding of the methylase to its own mRNA. It was shown by computer analysis that the same hexanucleotide (CCGCCC) is present 14 bp before the ribosome-binding site and in the C-1400 region of 16S rRNA, i.e., the region in which most of the aminoglycosides act. A deletion that removes the hexanucleotide before the gene fusion is not prone to negative autoregulation. This mode of regulation of the sgm gene ensures that enough methylase molecules protect the cell from the action of its own antibiotic. On the other hand, if all of the ribosomes are modified, Sgm methylase binds to its own mRNA in an autorepressive manner.

Bacterial Proteins↗

PubMed1994

Carbon Source Requirements for Exopolysaccharide Production by Lactobacillus casei CG11 and Partial Structure Analysis of the Polymer.

Exopolysaccharide production by Lactobacillus casei CG11 was studied in basal minimum medium containing various carbon sources (galactose, glucose, lactose, sucrose, maltose, melibiose) at concentrations of 2, 5, 10, and 20 g/liter. L. casei CG11 produced exopolysaccharides in basal minimum medium containing each of the sugars tested; lactose and galactose were the poorest carbon sources, and glucose was by far the most efficient carbon source. Sugar concentrations had a marked effect on polymer yield. Plasmid-cured Muc derivatives grew better in the presence of glucose and attained slightly higher populations than the wild-type strain. The values obtained with lactose were considerably lower for both growth and exopolysaccharide yield. The level of specific polymer production per cell obtained with glucose was distinctively lower for Muc derivatives than for the Muc strain. The polymer produced by L. casei CG11 in the presence of glucose was different from that formed in the presence of lactose. The polysaccharide produced by L. casei CG11 in basal minimum medium containing 20 g of glucose per liter had an intrinsic viscosity of 1.13 dl/g. It was rich in glucose (76%), which was present mostly as 2- or 3-linked residues along with some 2,3 doubly substituted glucose units, and in rhamnose (21%), which was present as 2-linked or terminal rhamnose; traces of mannose and galactose were also present.

Journal Article↗

PubMed1994

Mutational analysis of cat-86 gene expression controlled by lactococcal promoters in Lactococcus lactis subsp. lactis and Escherichia coli.

Promoters were cloned from the chromosomal DNA of Lactococcus lactis subsp. lactis NP4510 by using promoter-probe vector pGKV210. N-Methyl-N'-nitro-N-nitrosoguanidine-induced mutagenesis of L. lactis-(pBV413), with low-level expression of the cat-86 gene, resulted in enhanced expression. Subcloning and sequencing of the mutated plasmid designated pBV415 revealed that the mutation is located within the PstI-HindIII fragment containing the coding sequence of the cat-86 gene (the 10th CTG codon was replaced by a TTG; both code for leucine). A set of otherwise identical plasmids with four combinations of CTG and TTG codons at the 10th and 46th positions in the cat-86 gene were constructed by site-directed mutagenesis. These plasmids containing cat-86 derivatives displayed a significant variation in cat expression in L. lactis and E. coli. The data suggest that cat expression is dependent on the secondary structure of the cat mRNA. New cat-86 derivatives described here can be used in lactococci, in which they provide additional flexibility for promoter cloning.

Amino Acid Sequence↗

PubMed1994

Cloning of promoter-like sequences from Lactobacillus paracasei subsp. paracasei CG11 and their expression in Escherichia coli, Lactococcus lactis, and Lactobacillus reuteri.

Fragments of chromosomal DNA from Lactobacillus paracasei subsp. paracasei CG11 (formerly Lactobacillus casei CG11) capable of functioning as promoters were isolated using the broad host range, promoter-probe vector pGKV210. Five such fragments designated P61, P79, P80, P116, and P144 were completely sequenced and analyzed. Fragment P61 had the highest transcriptional efficiency in Escherichia coli and Lactobacillus reuteri whereas P80 was the most active in Lactococcus lactis. In general, the orders of the transcriptional strengths were almost identical in E. coli and Lactobacillus reuteri but different from that in Lactococcus lactis. Mapping of the 5' end of cat mRNA showed that different regions of fragments P79 and P144 were used as promoters in Lactococcus lactis than in E. coli and Lactobacillus reuteri. Analysis of these DNA sequences revealed that the putative -35 and -10 hexanucleotides resembled those of E. coli, Bacillus subtilis, and lactococci. The spacing between these two hexanucleotides and between the putative -10 hexanucleotide and the transcriptional start point (A residues predominated) ranged from 17 to 18 base pairs and from 5 to 7 base pairs, respectively. Each of the cloned Lactobacillus paracasei CG11 promoter-like fragments contained an AT-rich sequence upstream of the putative -35 region (from 60 to 73%).

Amino Acid Sequence↗

PubMed1993

A genetic study of apramycin-resistant mutants of Escherichia coli.

The apramycin-resistant mutants of E. coli were isolated spontaneously. Mutations, aprA and aprB, conferring resistance to apramycin were located at 72 min on the E. coli genetic map where most of ribosomal genes were mapped. One of the mutants, carrying the aprB mutation, has an altered translational fidelity expressed as severe restriction of amber suppressor activity in vivo. The chromosome location and altered translational fidelity indicate that the apramycin-resistant phenotype could be a consequence of an alteration of the ribosomal structure.

Chromosome Mapping↗

PubMed1993

Molecular analysis of the rolling-circle replicating plasmid pA1 of Lactobacillus plantarum A112.

Lactobacillus plantarum A112 has four different plasmids. Plus-origin-specific probes were used to determine that the smallest, cryptic plasmid, pA1 (2,820 bp), showed homology to the pE194 plasmid family. This subclass of plasmids uses the rolling-circle mode of replication. Subsequent analysis of plasmid pA1 demonstrated that it generates single-stranded DNA intermediates, and sequence analysis revealed that it contains three putative open reading frames (ORFs): ORF1, ORF2, and ORF3, which could encode proteins designated RepA (47 amino acids [aa]) and RepB (196 aa) and a protein of 103 aa, respectively. Two of these proteins, RepA (5.6 kDa) and RepB (26 kDa), were identified in in vitro transcription translation assays. The RepA protein contains a characteristic alpha-helix-turn-alpha-helix motif typical of DNA-binding proteins that act as DNA-binding repressors. The RepB protein shows a significant similarity with replication initiation proteins of the pE194 family of plasmids that use the rolling-circle mode of replication. Plasmid pA1 is able to replicate in Escherichia coli and Lactobacillus lactis subsp. lactis as well as in other L. plantarum strains.

Amino Acid Sequence↗

PubMed1992

Analysis of exopolysaccharide production by Lactobacillus casei CG11, isolated from cheese.

Exopolysaccharide-producing Lactobacillus casei CG11 was isolated from soft, white, homemade cheese. In basal minimal medium, it produces a neutral heteropolysaccharide consisting predominantly of glucose (about 75%) and rhamnose (about 15%). Plasmid curing experiments revealed that exopolysaccharide production by strain CG11 is linked to a plasmid approximately 30 kb in size.

Cheese↗

PubMed1992

Cloning and characterization of an aminoglycoside resistance determinant from Micromonospora zionensis.

The sisomicin-gentamicin resistance methylase (sgm) gene was isolated from Micromonospora zionensis and cloned in Streptomyces lividans. The sgm gene was expressed in Micromonospora melanosporea, where its own promoter was active, and also in Escherichia coli under the control of the lacZ promoter. The complete nucleotide sequence of 1,122 bp and a transcription start point were determined. The sequence contains an open reading frame that encodes a polypeptide of 274 amino acids. The methylation of 30S ribosomal subunits by Sgm methylase accounts adequately for all known resistance characteristics of M. zionensis, but expression of high-level resistance to hygromycin B is background dependent. A comparison of the amino acid sequence of the predicted Sgm protein with the deduced amino acid sequences for the 16S rRNA methylases showed extensive similarity of Grm and significant similarity to KgmB but not to KamB methylase.

Amino Acid Sequence↗

PubMed1991

Improved vector for promoter screening in lactococci.

Fragments of Lactococcus lactis subsp. lactis NP45 chromosomal DNA provided promoter activity in Escherichia coli when cloned into the promoter probe vector pGKV210. Only 13% of these recombinant plasmids promoted detectable cat-86 activity when transferred to L. lactis, i.e., expressed chloramphenicol resistance. In these promoter-containing versions of pGKV210, the cat-86 gene specifies chloramphenicol-inducible chloramphenicol acetyltransferase expression. This could be a limiting factor for cloning of promoters with lower activity in L. lactis. Therefore, we have constructed a new promoter probe vector, pBV5030, with the mutated version of the cat-86 gene, which is constitutively expressed when transcriptionally activated by the insertion of a promoter. We found that in L. lactis IL1403 the constitutively expressed cat-86 gene (on a pBV5030 derivative) has four times higher activity than the inducible version of the same gene (on a pGKV210 derivative) when both have the same promoter inserted upstream of the cat-86 gene. These results suggest that plasmid pBV5030 could be a more efficient vector for the cloning of promoters from lactococci.

Chloramphenicol O-Acetyltransferase↗

PubMed1991

Characterization of the Cell Wall-Bound Proteinase of Lactobacillus casei HN14.

Lactobacillus casei HN14, which was isolated from homemade cheese, produces an extracellular, cell wall-bound proteinase. The HN14 proteinase can be removed from the cell envelope by washing the cells in a Ca-free buffer. The activity of the crude proteinase extract is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the HN14 proteinase is similar to the lactococcal PI-type enzyme, since it hydrolyzes beta-casein only. Lactobacillus casei HN14 appeared to be plasmid free, which suggests that the proteinase gene is chromosomally located. Chromosomal DNA of this strain hybridizes with DNA probes Q1 (which contains a fragment of the prtM gene) and Q6 and Q92 (which contain fragments of the prtP gene); all three probes originated from the proteinase gene region of Lactococcus lactis subsp. cremoris Wg2. A restriction enzyme map of the proteinase region of Lactobacillus casei HN14 was constructed on the basis of hybridization experiments. Comparison of the restriction enzyme maps of the Lactobacillus casei HN14 proteinase gene region and those of lactococcal proteinase gene regions studied so far indicates that they are highly similar.

Journal Article↗

PubMed1991

Bacteriocin-Producing Strain of Lactococcus lactis subsp. diacitilactis S50.

Lactococcus lactis subsp. diacitilactis S50 produces a bacteriocin, designated bacteriocin S50, which has a narrow antibacterial spectrum. It was active only against Lactococcus species, including a nisin producer exhibiting a bactericidal effect. The activity of bacteriocin S50 was sensitive to proteases. It retained antimicrobial activity after being heated to 100 degrees C for up to 60 min and in the pH range 2 to 11.

Journal Article↗

PubMed1990

Altered translational fidelity of a Salmonella typhimurium LT2 mutant resistant to the aminoglycoside antibiotic neamine.

A neamine-resistant mutant of Salmonella typhimurium LT2 with altered translational fidelity was isolated. The phenomenon was expressed in severe restriction of amber suppressor activity in vivo as well as in decreased misreading of poly(U) RNA in vitro. The mutation conferring resistance to neamine was mapped at 72 min on the Salmonella genetic map, where some of the ribosomal genes have already been mapped. This location indicates that the neamine-resistant phenotype as well as an altered translational fidelity could be a consequence of an alteration of the ribosomal structure.

Chromosome Mapping↗