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A Palva

Publications and source records attributed to A Palva.

At least 37 records · Page 2Linked to original sources
PubMed1997

High level heterologous protein production in Lactococcus and Lactobacillus using a new secretion system based on the Lactobacillus brevis S-layer signals.

A secretion cassette, based on the expression and secretion signals of a S-layer protein (SlpA) from Lactobacillus brevis, was constructed. E. coli beta-lactamase (Bla) was used as the reporter protein to determine the functionality of the S-layer signals for heterologous expression and secretion in Lactococcus lactis, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus gasseri and Lactobacillus casei using a low-copy-number plasmid derived from pGK12. In all hosts tested, the bla gene was expressed under the slpA signals and all Bla activity was secreted to the culture medium. The Lb. brevis S-layer promoters were very efficiently recognized in L. lactis, Lb. brevis and Lb. plantarum, whereas in Lb. gasseri the slpA promoter region appeared to be recognized at a lower level and in Lb. casei the level of transcripts was below the detection limit. The production of Bla was mainly restricted to the exponential phase of growth. The highest yield of Bla was obtained with L. lactis and Lb. brevis. Without pH control, substantial degradation of Bla occurred during prolonged cultivations with all lactic acid bacteria (LAB) tested. When growing L. lactis and Lb. brevis under pH control, the Bla activity could be stabilized also at the stationary phase. L. lactis produced up to 80 mg/l of Bla which to our knowledge represents the highest amount of a heterologous protein secreted by LAB so far. The short production phase implied a very high rate of secretion with a calculated value of 5 x 10(5) Bla molecules/cell per h. Such a high rate was also observed with Lb. plantarum, whereas in Lb. brevis the competition between the wild type slpA gene and the secretion construct probably lowered the rate of Bla production. The results obtained indicate wide applicability of the Lb. brevis slpA signals for efficient protein production and secretion in LAB.

Base Sequence↗
PubMed1997

Molecular genetic characterization of the L-lactate dehydrogenase gene (ldhL) of Lactobacillus helveticus and biochemical characterization of the enzyme.

The Lactobacillus helveticus L-(+)-lactate dehydrogenase (L-LDH) gene (ldhL) was isolated from a lambda library. The nucleotide sequence of the ldhL gene was determined and shown to have the capacity to encode a protein of 323 amino acids (35.3 kDa). The deduced sequence of the 35-kDa protein revealed a relatively high degree of identity with other lactobacillar L-LDHs. The highest identity (80.2%) was observed with the Lactobacillus casei L-LDH. The sizes and 5' end analyses of ldhL transcripts showed that the ldhL gene is a monocistronic transcriptional unit. The expression of ldhL, studied as a function of growth, revealed a high expression level at the logarithmic phase of growth. The ldhL gene is preceded by two putative -10 regions, but no corresponding -35 regions could be identified. By primer extension analysis, the ldhL transcripts were confirmed to be derived from the -10 region closest to the initiation codon. However, upstream of these regions additional putative -10/-35 regions could be found. The L-LDH was overexpressed in Escherichia coli and purified to homogeneity by two chromatographic steps. The purified L-LDH was shown to be a nonaliosteric enzyme, and amino acid residues involved in allosteric regulation were not conserved in L. helveticus L-LDH. However, a slight enhancement of enzyme activity was observed in the presence of fructose 1,6-diphosphate, particularly at neutral pH. A detailed enzymatic characterization of L-LDH was performed. The optimal reaction velocity was at pH 5.0, where the kinetic parameters K(m), and Kcat for pyruvate were 0.25 mM and 643 S-1, respectively.

Amino Acid Sequence↗
PubMed1997

In vivo expression of the Lactobacillus brevis S-layer gene.

Lactobacillus brevis possesses a surface layer protein (SlpA) with tightly regulated synthesis. The slpA gene is expressed by two adjacent promoters, P1 and P2. The level of P2-derived transcripts was approximately 10 times higher than that of P1-derived transcripts throughout the entire growth of L. brevis. The half-lives of slpA transcripts were shown to be exceptionally long (14 min).

Bacterial Outer Membrane Proteins↗
PubMed1996

Molecular characterization, over-expression and purification of a novel dipeptidase from Lactobacillus helveticus.

A dipeptidase gene (pepD) from an industrial Lactobacillus helveticus strain was isolated by colony hybridization. An open reading frame (ORF) of 1422 base pairs (bp) with a coding capacity for a 53.5-kDa protein (PepD) was identified. The ORF was preceded by a typical prokaryotic promoter region, and an inverted repeat structure with delta G of -51.0 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of the 53.5-kDa protein revealed no marked homologies when compared to the data bases of EMBL and SWISS-PROT. The 5'end of the 1.6-kb pepD transcript was determined both by a conventional primer extension method and using an automated sequencer. pepD was found to be maximally expressed at late exponential growth. The pepD gene was cloned into an expression vector to over-produce PepD in Escherichia coli JM105. Purification of PepD to homogeneity was achieved using three chromatographic steps. PepD was able to hydrolyze a number of dipeptides with the exception of those containing a proline residue. Optimal PepD activity was observed at pH 6.0 and 55 degrees C. The enzyme was inhibited by p-hydroxymercuribenzoate and reactivated by dithiothreitol whereas ethylenediaminetetraacetate had no inhibitory effect on PepD. The enzymatic properties of PepD suggest that it represents a novel dipeptidase type among lactic acid bacteria.

Amino Acid Sequence↗
PubMed1996

An operon from Lactobacillus helveticus composed of a proline iminopeptidase gene (pepI) and two genes coding for putative members of the ABC transporter family of proteins.

A proline iminopeptidase gene (pepI) of an industrial Lactobacillus helveticus strain was cloned and found to be organized in an operon-like structure of three open reading frames (ORF1, ORF2 and ORF3). ORF1 was preceded by a typical prokaryotic promoter region, and a putative transcription terminator was found downstream of ORF3, identified as the pepI gene. Using primer-extension analyses, only one transcription start site, upstream of ORF1, was identifiable in the predicted operon. Although the size of mRNA could not be judged by Northern analysis either with ORF1-, ORF2- or pepI-specific probes, reverse transcription-PCR analyses further supported the operon structure of the three genes. ORF1, ORF2 and ORF3 had coding capacities for 50.7, 24.5 and 33.8 kDa proteins, respectively. The ORF3-encoded PepI protein showed 65% identity with the PepI proteins from Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis. The ORF1-encoded protein had significant homology with several members of the ABC transporter family but, with two distinct putative ATP-binding sites, it would represent an unusual type among the bacterial ABC transporters. ORF2 encoded a putative integral membrane protein also characteristic of the ABC transporter family. The pepI gene was overexpressed in Escherichia coli. Purified PepI hydrolysed only di and tripeptides with proline in the first position. Optimum PepI activity was observed at pH 7.5 and 40 degrees C. A gel filtration analysis indicated that PepI is a dimer of M(r) 53,000. PepI was shown to be a metal-independent serine peptidase having thiol groups at or near the active site. Kinetic studies with proline-p-nitroanilide as substrate revealed Km and Vmax values of 0.8 mM and 350 mmol min-1 mg-1, respectively, and a very high turnover number of 135,000 s-1.

ATP-Binding Cassette Transporters↗
PubMed1995

Sequence analysis, distribution and expression of an aminopeptidase N-encoding gene from Lactobacillus helveticus CNRZ32 [gene 155 (1995) 89-93].

Lactobacillus (Lb.) helveticus CNRZ32 possesses a 97-kDa metalloenzyme with aminopeptidase activity (PepN; EC 3.4.11.2). A 3.8-kb fragment encoding PepN was cloned into pIL253 and designated pSUW34. Transformation of Lactococcus (Lc.) lactis LM0230 with pSUW34 resulted in > 180-fold increase in general aminopeptidase (AP) activity using L-lysine-p-nitroanilide. Southern hybridization was conducted to determine the distribution of homology to the CNRZ32 pepN gene among lactic-acid bacteria (LAB). Hybridization was observed with strains of lactobacilli, pediococci, leuconostoc, streptococci and lactococci. The pepN gene was sequenced and found to encode a protein containing 844 amino acid (aa) residues. A comparison of Lb. helveticus CNRZ32 pepN to Lb. delbrueckii ssp. lactis DSM7290 pepN indicated 69.5% nucleotide (nt) identity and 71.8% aa identity, while comparison to pepN from Lc. lactis ssp. cremoris MG1363 indicated 61.1% nt identity and 49.2% aa identity. Alignment of peptidase aa sequences of LAB, Escherichia coli, yeast and mammalian origin display homology in the zinc-binding domain, as well as a conserved region upstream from the putative active site.

Amino Acid Sequence↗
PubMed1995

Sequence analysis, distribution and expression of an aminopeptidase N-encoding gene from Lactobacillus helveticus CNRZ32.

Lactobacillus (Lb.) helveticus CNRZ32 possesses a 97-kDa metalloenzyme with aminopeptidase activity (PepN; EC 3.4.11.2). A 3.8-kb fragment encoding PepN was cloned into pIL253 and designated pSUW34. Transformation of lactococcus (Lc.) lactis LM0230 with pSUW34 resulted in > 180-fold increase in general aminopeptidase (AP) activity using L-lysine-p-nitroanilide. Southern hybridization was conducted to determine the distribution of homology to the CNRZ32 pepN gene among lactic-acid bacteria (LAB). Hybridization was observed with strains of lactobacilli, pediococci, leuconostoc, streptococci and lactococci. The pepN gene was sequenced and found to encode a protein containing 844 amino acid (aa) residues. A comparison of Lb. helveticus CNRZ32 pepN to Lb. delbrueckii ssp. lactis DSM7290 pepN indicated 69.5% nucleotide (nt) identity and 71.8% aa identity, while comparison to pepN from Lc. lactis ssp. cremoris MG1363 indicated 61.1% nt identity and 49.2% aa identity. Alignment of peptidase aa sequences of LAB, Escherichia coli, yeast and mammalian origin display homology in the zinc-binding domain, as well as a conserved region upstream from the putative active site.

Amino Acid Sequence↗
PubMed1995

An X-prolyl dipeptidyl aminopeptidase (pepX) gene from Lactobacillus helveticus.

The X-prolyl dipeptidyl aminopeptidase gene (pepX) of an industrially used Lactobacillus helveticus strain has been detected by nucleic acid hybridization, cloned, characterized and sequenced. One ORF of 2379 bp with coding capacity for a 90.6 kDa protein (PepX) was found. The ORF was preceded by a typical prokaryotic promoter region. An inverted repeat structure with delta G of -84.1 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of the 90.6 kDa protein showed 49.3, 49.4 and 77.7% homology with the PepX proteins from Lactococcus lactis subsp. lactis, Lc. lactis subsp. cremoris and Lactobacillus delbrueckii subsp. lactis, respectively. Northern blotting revealed a 2.6 kb transcript and one transcription start site was identified via primer extension analysis using an A.L.F. sequencer. In a bioreactor study, the expression of pepX in Lb. helveticus was studied as a function of growth. Transcription of pepX was typical of exponential growth phase expression. The pepX gene has been cloned into pKK223-3 and expressed at a high level in Escherichia coli JM105. PepX was purified to homogeneity by ion-exchange and hydrophobic interaction chromatography. Optimum PepX activity was observed at pH 6.5 and 45 degrees C. According to gel filtration analysis, PepX is a dimer of 165 kDa. The enzyme was inactivated by heavy metal ions such as Cu2+, Cd2+ and Zn2+. EDTA and 1,10-phenanthroline did not decrease PepX activity significantly. It was completely inhibited by p-hydroxymercuribenzoate and reactivated by adding DTT, and strongly inhibited by PMSF. PepX is thus a metal-independent serine peptidase having functional sulfhydryl groups at or near the active site.

Amino Acid Sequence↗
PubMed1994

Characterization and expression of the pepN gene encoding a general aminopeptidase from Lactobacillus helveticus.

An aminopeptidase N (pepN) gene was detected by DNA hybridization from an industrially important Lactobacillus helveticus strain using part of the L. helveticus CNRZ32 pepN gene as the probe. One of five hybridization positive clones was characterized in more detail. A subcloned 3.7 kb fragment, positive in hybridization and encoding aminopeptidase activity, was sequenced and analyzed. Only one open reading frame (ORF) of 2532 base pairs with a coding capacity for a 95.9 kDa protein could be found. The deduced amino acid sequence of the 95.9 kDa protein showed homology to PepN proteins from other lactic acid bacteria and carried the conserved catalytic and zinc binding sites of the neutral zinc metallo-peptidase family confirming the identity of the pepN gene. A 2.75 kb transcript and two transcription start sites were identified with mRNA analyses. Expression of pepN in L. helveticus, studied as the function of growth, revealed a high level of pepN transcripts throughout the growth, in contrast to the steady state levels of other peptidase mRNAs from L. helveticus analyzed in our laboratory.

Amino Acid Sequence↗
PubMed1994

Characterization and expression of the Lactobacillus helveticus pepC gene encoding a general aminopeptidase.

An aminopeptidase C gene (pepC) was detected by nucleic acid hybridization from an industrially important Lactobacillus helveticus strain. Three hybridization positive clones were isolated from a gene library of this L. helveticus strain, and one of them was characterized in more detail. Deletion mapping localized the hybridization positivity into a 2.8-kb fragment, which also encoded aminopeptidase activity. This fragment was sequenced and two open reading frames (ORF1 and 2) of 1347 and 840 base pairs were identified. The ORF1 was preceded by a typical prokaryotic promoter region, and an inverted repeat structure with delta G of -49.0 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of ORF1, with an encoding capacity for a 51.4-kDa protein, was shown to share 48.3% and 98.0% identities with the PepC proteins from Lactococcus lactis and L. helveticus CNRZ32, respectively, thus confirming that ORF1 codes for an aminopeptidase C. mRNA size analyses revealed 1.7-kb and 2.7-kb transcripts in Northern blot with the pepC-specific probe. A further analysis with the pepC- and ORF2-specific probes showed that downstream ORF2 is co-transcribed with the pepC gene at the exponential phase of growth whereas, at the stationary growth phase, transcripts derived from the pepC promoter were below the detection limit, and the ORF2 was expressed by its own promoter. The 5' end mapping of the pepC transcripts with primer extension revealed one transcription start site suggesting a new position for the pepC promoter region when compared to that predicted for the L. helveticus CNRZ32 pepC gene. Expression of pepC was also studied in L. helveticus as the function of growth in a bioreactor study. Transcription of pepC was typical to exponential growth phase expression. The level of total thiol-aminopeptidase activity, however, remained nearly constant throughout the stationary growth phase.

Amino Acid Sequence↗
PubMed1992

S-layer protein gene of Lactobacillus brevis: cloning by polymerase chain reaction and determination of the nucleotide sequence.

The surface (S)-layer protein of Lactobacillus brevis was isolated, purified, and characterized. The S-layer protein is the major protein of the cell, with an apparent molecular mass of 46 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunogold electron microscopy with polyclonal antiserum against the isolated 46-kDa protein was used to confirm the surface location of this protein. N-terminal amino acid sequences of the intact 46-kDa protein and its tryptic peptides were determined. The gene of the S-layer protein was amplified from the genome of L. brevis by polymerase chain reaction with oligonucleotides, synthesized according to the N-terminal amino acid sequences, as primers. The polymerase chain reaction fragments containing the entire S-layer gene and its regulatory regions were sequenced. Nucleic acid sequence analysis revealed one open reading frame with a capacity to encode a protein of 48,159 Da. From the regulatory region of the gene, two subsequent promoters and a ribosome binding site, showing typical features of prokaryotic consensus sequences, were found. The coding region contained a characteristic gram-positive-type signal peptide of 30 amino acids. Removal of the signal peptide results in a polypeptide of 435 amino acids, which is in excellent agreement with the size of the S-layer protein determined by SDS-PAGE. The size and the 5' end analyses of the S-layer transcripts confirmed the monocistronic nature of the S-layer operon and the functionality of the two promoters found.

Amino Acid Sequence↗
PubMed1992

Effect of positioning of the feet in posturography.

Static posturography has been developed from a basic research test to a widely used clinical tool for evaluating dizzy patients. Before any actual standardization can be achieved, however, several aspects of the test situations have to be evaluated, including the position of the feet. The quantitative significance of the standing position in posturographic measurements was evaluated with healthy volunteers studied under visual and nonvisual conditions, using 4 foot positions: heels together with the toes 30 degrees apart or at an angle of the volunteer's own choice, and the feet parallel and either 0 or 10 cm apart were studied separately. Each measurement was characterized in terms of 5 parameters (body sway velocity, vibration-induced shift of centre of force in anteroposterior and lateral directions, and maximum displacement of centre of force in the same directions). Body sway velocities were smallest when the feet were parallel and 10 cm apart. Although the position chosen by the subject was usually more stable than that with the toes 30 degrees apart, the difference was nonsignificant. According to our results, the standing position is not crucial in posturographic measurements provided that the distance between the heels is determined, and the subject can just as well choose the angle between the feet if the heels are kept together.

Adult↗
PubMed1990

Production and secretion of pertussis toxin subunits in Bacillus subtilis.

Pertussis toxin (PT) is a major component of today's acellular whooping cough vaccines. The use of acellular vaccines is predicted to increase sharply in the near future. There is therefore a need to produce PT in a way that makes its purification as easy as possible. Our approach was to express all five PT subunits individually in Bacillus subtilis. We have used vectors containing the promoter and signal sequences of the alpha-amylase gene of Bacillus amyloliquefaciens followed by an insert encoding the appropriate PT-subunit. All PT-subunits were secreted and found in the culture supernatant. The level of expression varied considerably: S1 and S5 were produced in large quantities whereas much smaller amounts of S2, S3 and S4 were found. The subunits were also present in the membrane fraction of the respective strains.

Amino Acid Sequence↗
PubMed1990

Secretory S complex of Bacillus subtilis: sequence analysis and identity to pyruvate dehydrogenase.

We have cloned the operon coding for the Bacillus subtilis S complex, which has been proposed to be a component in protein secretion machinery. A lambda gt10 library of B. subtilis was screened with antiserum directed against the Staphylococcus aureus membrane-bound ribosome protein complex, which is homologous to the B. subtilis S complex. Two positive overlapping lambda clones were sequenced. The S-complex operon, 5 kilobases in size, was shown to contain four open reading frames and three putative promoters, which are located upstream of the first, the third, and the last gene. The four proteins encoded by the operon are 42, 36, 48, and 50 kilodaltons in size. All of these proteins were recognized by antisera separately raised against each protein of the S. aureus membrane-bound ribosome protein and B. subtilis S complexes, thus verifying the S-complex identity of the lambda clones. Sequence analysis revealed that all four proteins of the B. subtilis S complex are homologous to the four subunits of the human pyruvate dehydrogenase (PDH). Also, the N terminus of the 48-kilodalton protein was found to have 70% amino acid identity with the N-terminal 211 amino acids, determined so far, from the E2 subunit of B. stearothermophilus PDH. Furthermore, chromosomal mapping of the S-complex operon gave a linkage to a marker gene located close to the previously mapped B. subtilis PDH genes. Thus, the S complex is evidently identical to the B. subtilis PDH, which has been shown to contain four subunits with molecular weights very similar to those of the S complex. Therefore, we propose that the S complex is not a primary component of protein secretion.

Amino Acid Sequence↗
PubMed1988

Long-term presence of a denture fragment in the airway (a report of two cases).

Two cases are reported in which a denture fragment was lodged in the bronchus for a period of six years. Bronchial asthma which did not worsen immediately after inhalation and other misleading factors made the diagnosis more difficult in the first case, whilst the respiratory symptoms in the second case appeared only 4 years after inhalation. Haemoptysis 2 years later led to a bronchoscopy and the removal of the foreign body.

Aged↗
PubMed1988

Quantification of alpha-amylase mRNA in Bacillus subtilis by nucleic acid sandwich hybridization.

A novel hybridization test for quantification of mRNA from bacterial cells lysed directly in the culture medium was developed and optimized. The method uses two adjacent probes from a DNA fragment of interest in a sandwich hybridization. An unlabeled probe is immobilized on a solid support to capture homologous nucleic acids in the test solution. Hybrid detection is performed with the other, labeled, probe, which can bind to the filter only as a sample-mediated hybrid. We used the Bacillus amyloliquefaciens alpha-amylase gene and its mRNA as models for characterizing gene expression in Bacillus subtilis. To confirm the specificity of mRNA hybridization, nondenatured nucleic acid samples were allowed to react simultaneously with two filters: the mRNA-specific filter containing anti-sense single-stranded DNA and the background filter containing single-stranded DNA of the mRNA sense, respectively. We also developed a rapid enzymatic lysis procedure for B. subtilis, allowing complete degradation of late stationary phase cells grown in rich culture medium, while retaining mRNA molecules of high integrity. The applicability of this rapid lysis procedure and the hybridization method for mRNA analyses in long-term fermentations was demonstrated.

Bacillus subtilis↗
PubMed1988

Acoustic neuroma surgery in northern Finland.

A series of 40 patients with acoustic neuromas were operated on using the suboccipital or the translabyrinthine approach. Some patients were operated on using both routes. The series extends back to 1977 and contain predominantly large tumours. There was no mortality in the translabyrinthine series of 14 patients. In the suboccipitally treated series of 29 patient, 3 were previously treated using translabyrinthine approach. In this series there was one death directly due to sequelae of neuroma extirpation and in another, additional causes were present. Preoperative facial pareses were frequent. There were several additional pareses in the translabyrinthine series and also several additional in the suboccipital series. Facial pareses were operated on by a plastic surgeon and they did not markedly detract from working capacity. Only 2 patients in the total series had small tumours and useful preoperative hearing. Cochlear function was preserved in them.

Adult↗