Increased breath naphthalene in children with asthma and wheeze of the All Age Asthma Cohort (ALLIANCE).

Exhaled breath contains numerous volatile organic compounds (VOCs) known to be related to lung disease like asthma. Its collection is non-invasive, simple to perform and therefore an attractive method for the use even in young children. We analysed breath in children of the multicenter All Age Asthma Cohort (ALLIANCE) to evaluate if 'breathomics' have the potential to phenotype patients with asthma and wheeze, and to identify extrinsic risk factors for underlying disease mechanisms. A breath sample was collected from 142 children (asthma: 51, pre-school wheezers: 55, healthy controls: 36) and analysed using gas chromatography-mass spectrometry (GC/MS). Children were diagnosed according to Global Initiative for Asthma guidelines and comprehensively examined each year over up to seven years. Forty children repeated the breath collection after 24 or 48 months. Most breath VOCs differing between groups reflect the exposome of the children. We observed lower levels of lifestyle-related VOCs and higher levels of the environmental pollutants, especially naphthalene, in children with asthma or wheeze. Naphthalene was also higher in symptomatic patients and in wheezers with recent inhaled corticosteroid use. No relationships with lung function or TH2 inflammation were detected. Increased levels of naphthalene in asthmatics and wheezers and the relationship to disease severity could indicate a role of environmental or indoor air pollution for the development or progress of asthma. Breath VOCs might help to elucidate the role of the exposome for the development of asthma. The study was registered at ClinicalTrials.gov (NCT02496468).

Impact of different sugars and glycosyltransferases on the assertiveness of Latilactobacillus sakei in raw sausage fermentations.

Latilactobacillus sakei comprises a biodiversity of strains, which display different assertiveness upon their application as starter cultures in raw sausage fermentation. While the assertiveness of winning partner strains has been referred to competitive exclusion based on genomic settings enabling occupation of multiple niches of the sausage habitat, single strain assertiveness of L. sakei remained unexplained. In this study we assessed the impact of the expression of a glycosyltransferase enabling the production of a glucan from sucrose to the assertiveness of L. sakei TMW 1.411, which expresses a plasmid-encoded glycosyltransferase (gtf). In a sausage fermentation model wild type L. sakei TMW 1.411 and its plasmid-cured mutant were employed in competition with each other and with other Latilactobacillus sakei strains. To differentiate any effects resulting from general sugar utilization from those of glucan formation, the experiments were carried out with glucose, fructose, and sucrose, respectively. It was shown that the type of sugar affects the individual strains behaviour, and that the wild type was more competitive than the mutant in the presence of any of these sugars. In direct competition between wild type and mutant, a clear competitive advantage could also be demonstrated for the strain possessing the plasmid with the glycosyltransferase. Since this competitive advantage was observed with all sugars, not just sucrose, and Gtf expression has been shown as independent of the employed sugar, it is suggested that possession of the gtf gene-carrying plasmid confers a competitive advantage. It appears that the Gtf contributes to competitive exclusion and the establishment of colonization resistance, to a larger extent by an adhesive functionality of the Gtf on the cellular surface than by the production of glucan. Hence, gtf genes can be used as a possible additional marker for the selection of assertive L. sakei starter strains in sausage fermentation.

Comparative genome analysis, predicted lifestyle and antimicrobial strategies of Lactococcus carnosus and Lactococcus paracarnosus isolated from meat.

Strains of the psychrotrophic bacterium Lactococcus piscium have gained increasing attention as potentially bioprotective cultures due to their assertiveness against fish and meat spoilage bacteria as well as pathogenic bacteria. Recently, we have described two novel species within the genus Lactococcus (Lc.) namely Lc. carnosus (TMW 2.1612T) and Lc. paracarnosus (TMW 2.1615T) isolated from modified atmosphere packaged meat. Within this study, we compared the genomes of two Lc. carnosus strains, two Lc. paracarnosus strains and 16 Lc. piscium strains from our laboratory and five publicly available genomes previously affiliated to the species Lc. piscium. Our phylogenetic analysis supports reclassification of 20 of the strains to either Lc. carnosus or Lc. paracarnosus, so far limiting the Lc. piscium type strain (DSM 6634T) as sole representative of this species. Comparative genomics approach was conducted to predict underlying mechanisms involved in interspecies competition strategies of Lc. carnosus and Lc. paracarnosus against meat spoilers and predict their lifestyle in meat environments. In general, strains of the three species were highly similar regarding metabolic pathways for most of the relevant meat-derived substrates. In silico analyses enabled prediction of homolactic hexose fermentation by Lc. carnosus, Lc. paracarnosus and Lc. piscium. Further, genes required for the heterofermentative metabolism of hexoses and pentoses were only found in the Lc. pisicum type strain (DSM 6634T). We predict a low spoilage potential for Lc. carnosus and Lc. paracarnosus strains. No genes for decarboxylation of amino acids yielding biogenic amines were found in the genomes. Regarding their antimicrobial mechanisms against spoilers, we found a strain-specific putative polymorphic toxin system predictively delivered by the type VIIb secretion system, enabling cell-to-cell contact-dependent growth inhibition. Furthermore, we found additional genes predictively involved to the suppression of spoilers within the food microbiome (prophages, lytic domains, bacteriocins, metabolites).

Fermentation profile, cholesterol-reducing properties and chemopreventive potential of ß-glucans from Levilactobacillus brevis and Pediococcus claussenii - a comparative study with ß-glucans from different sources.

The aim of the present study was to investigate whether ß-glucans obtained from the lactic acid bacteria (LAB) Levilactobacillus (L.) brevis and Pediococcus (P.) claussenii exhibit similar physiological effects such as cholesterol-binding capacity (CBC) as the structurally different ß-glucans from oat, barley, and yeast as well as curdlan. After in vitro fermentation, fermentation supernatants (FSs) and/or -pellets (FPs) were analyzed regarding the concentrations of short-chain fatty acids (SCFAs), ammonia, bile acids, the relative abundance of bacterial taxa and chemopreventive effects (growth inhibition, apoptosis, genotoxicity) in LT97 colon adenoma cells. Compared to other glucans, the highest CBC was determined for oat ß-glucan (65.9 ± 8.8 mg g-1, p < 0.05). Concentrations of SCFA were increased in FSs of all ß-glucans (up to 2.7-fold). The lowest concentrations of ammonia (down to 0.8 ± 0.3 mmol L-1) and bile acids (2.5-5.2 µg mL-1) were detected in FSs of the ß-glucans from oat, barley, yeast, and curdlan. The various ß-glucans differentially modulated the relative abundance of bacteria families and reduced the Firmicutes/Bacteroidetes ratio. Treatment of LT97 cells with the FSs led to a significant dose-dependent growth reduction and increase in caspase-3 activity without exhibiting genotoxic effects. Though the different ß-glucans show different fermentation profiles as well as cholesterol- and bile acid-reducing properties, they exhibit comparable chemopreventive effects.

Acid stress response of Staphylococcus xylosus elicits changes in the proteome and cellular membrane.

AIMS: Coagulase-negative Staphylococcus xylosus strains are used as starter organisms for sausage fermentation. As those strains have to cope with low pH-values during fermentation, the aim of this study was to identify the acid adaptation mechanisms of S. xylosus TMW 2.1523 previously isolated from salami. METHODS AND RESULTS: A comparative proteomic study between two different acid tolerant mutants was performed. Therefore, both S. xylosus mutants were grown pH-static under acid stress (pH 5·1) and reference conditions (pH 7·0). Proteomic data were supported by metabolite and cell membrane lipid analysis. Staphylococcus xylosus acid stress adaptation is mainly characterized by a metabolic change towards neutral metabolites, enhanced urease activity, reduced ATP consumption, an increase in membrane fluidity and changes in the membrane thickness. CONCLUSION: This study corroborates mechanisms as previously described for other Gram-positive bacteria. Additionally, the adjustment of membrane structure and composition in S. xylosus TMW 2.1523 play a prominent role in its acid adaptation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates for the first time changes in the membrane lipid composition due to acid stress adaptation in staphylococci.

Monitoring of assertive Lactobacillus sakei and Lactobacillus curvatus strains using an industrial ring trial experiment.

AIMS: In a previous study, we used a 5-day fermenting sausage model to characterize assertiveness of Lactobacillus curvatus and Lactobacillus sakei starter strains towards employ autochthonous contaminants. In this work, we probed those findings and their transferability to real sausage fermentation including the drying process in an industrial ring trial experiment. METHODS AND RESULTS: Raw fermented sausages ('salami') were produced with three L. curvatus and four L. sakei strains as starter cultures in cooperation with three manufacturers from Germany. We monitored pH, water activity and microbiota dynamics at strain level over a total fermentation and ripening time of 21 days by MALDI-TOF-MS identification of isolates. The principal behaviour of the strains in real sausage fermentations was the same as that one observed in the 5-day model system delineating single strain assertiveness of a bacteriocin producer from co-dominance of strains. CONCLUSIONS: The water activity decrease, which is concomitant with the sausage ripening process has only limited impact on the assertiveness and survival of the starter strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of a 5-day model can provide insight in the assertiveness of a specific starter strain in sausage fermentation.

An adapted isolation procedure reveals Photobacterium spp. as common spoilers on modified atmosphere packaged meats.

The genus Photobacterium comprises species of marine bacteria, commonly found in open-ocean and deep-sea environments. Some species (e.g. Photobacterium phosphoreum) are associated with fish spoilage. Recently, culture-independent studies have drawn attention to the presence of photobacteria on meat. This study employed a comparative isolation approach of Photobacterium spp. and aimed to develop an adapted isolation procedure for recovery from food samples, as demonstrated for different meats: Marine broth is used for resuspending and dilution of food samples, followed by aerobic cultivation on marine broth agar supplemented with meat extract and vancomycin at 15°C for 72 h. Identification of spoilage-associated microbiota was carried out via Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry using a database supplemented with additional mass spectrometry profiles of Photobacterium spp. This study provides evidence for the common abundance of multiple Photobacterium species in relevant quantities on various modified atmosphere packaged meats. Photobacterium carnosum was predominant on beef and chicken, while Photobacterium iliopiscarium represented the major species on pork and Photobacterium phosphoreum on salmon, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates highly frequent isolation of multiple photobacteria (Photobacterium carnosum, Photobacterium phosphoreum, and Photobacterium iliopiscarium) from different modified-atmosphere packaged spoiled and unspoiled meats using an adapted isolation procedure. The abundance of photobacteria in high numbers provides evidence for the hitherto neglected importance and relevance of Photobacterium spp. to meat spoilage.

Comparative phylobiomic analysis of the bacterial community of water kefir by 16S rRNA gene amplicon sequencing and ARDRA analysis.

AIMS: The aim of this study was to analyse the bacterial microbiota of water kefir using culture-independent methods. METHODS AND RESULTS: We compared four water kefirs of different origins using 16S rDNA amplicon sequencing and ARDRA. The microbiota consisted of different proportions of the genera Lactobacillus (Lact.), Leuconostoc (Leuc.), Acetobacter (Acet.) and Gluconobacter. Surprisingly, varying but consistently high numbers of sequences representing members of the genus Bifidobacterium (Bif.) were found in all kefirs. Whereas part of the bifidobacterial sequences could be assigned to Bifidobacterium psychraerophilum, a majority of sequences identical to each other could not be assigned to any known species. A nearly full-length sequence of the latter exhibited a beyond-species similarity (96.4%) with the sequence from the closest relative species Bif. psychraerophilum. A Bifidobacterium-specific ARDRA analysis reflected the abundance of the novel Bifidobacterium species by revealing its unique MboI restriction profile. Attempts to isolate the bifidobacteria were successful for Bif. psychraerophilum only. CONCLUSIONS: The complexity of the water kefir microbiota has been underestimated in previously studies. The occurrence of bifidobacteria as part of the consortium is novel. SIGNIFICANCE AND IMPACT OF THE STUDY: These data give new insights into the understanding of the complexity of food fermentations and underline the need for approaches detecting noncultivable organisms.

MiR-30e and miR-181d control radial glia cell proliferation via HtrA1 modulation.

The precise mechanisms by which microRNAs (miRNAs) contribute to the dynamic regulation of gene expression during the forebrain development are still partly elusive. Here we show that the depletion of miRNAs in the cerebral cortex and hippocampus, via genetic inactivation of Dicer after the onset of forebrain neurogenesis, profoundly impairs the morphological and proliferative characteristics of neural stem and progenitor cells. The cytoarchitecture and self-renewal potential of radial glial (RG) cells located within the cerebral cortex and the hippocampus were profoundly altered, thus causing a significant derangement of both the post natal dorsal sub-ventricular zone and the dentate gyrus. This effect was attributed to the High-temperature requirement A serine peptidase 1 (HtrA1) gene product whose overexpression in the developing forebrain recapitulated some of the aspects of the Dicer(-/-) phenotype. MiR-30e and miR-181d were identified as posttranscriptional negative regulators of HtrA1 by binding to its 3' untranslated region. In vivo overexpression of miR-30e and miR-181d in Dicer(-/-) forebrain rescued RG proliferation defects.

HtrA1-dependent proteolysis of TGF-beta controls both neuronal maturation and developmental survival.

Transforming growth factor-beta (TGF-beta) signalling controls a number of cerebral functions and dysfunctions including synaptogenesis, amyloid-beta accumulation, apoptosis and excitotoxicity. Using cultured cortical neurons prepared from either wild type or transgenic mice overexpressing a TGF-beta-responsive luciferase reporter gene (SBE-Luc), we demonstrated a progressive loss of TGF-beta signalling during neuronal maturation and survival. Moreover, we showed that neurons exhibit increasing amounts of the serine protease HtrA1 (high temperature responsive antigen 1) and corresponding cleavage products during both in vitro neuronal maturation and brain development. In parallel of its ability to promote degradation of TGF-beta1, we demonstrated that blockage of the proteolytic activity of HtrA1 leads to a restoration of TGF-beta signalling, subsequent overexpression of the serpin type -1 plasminogen activator inhibitor (PAI-1) and neuronal death. Altogether, we propose that the balance between HtrA1 and TGF-beta could be one of the critical events controlling both neuronal maturation and developmental survival.

High pressure-sensitive gene expression in Lactobacillus sanfranciscensis.

Lactobacillus sanfranciscensis is a Gram-positive lactic acid bacterium used in food biotechnology. It is necessary to investigate many aspects of a model organism to elucidate mechanisms of stress response, to facilitate preparation, application and performance in food fermentation, to understand mechanisms of inactivation, and to identify novel tools for high pressure biotechnology. To investigate the mechanisms of the complex bacterial response to high pressure we have analyzed changes in the proteome and transcriptome by 2-D electrophoresis, and by microarrays and real time PCR, respectively. More than 16 proteins were found to be differentially expressed upon high pressure stress and were compared to those sensitive to other stresses. Except for one apparently high pressure-specific stress protein, no pressure-specific stress proteins were found, and the proteome response to pressure was found to differ from that induced by other stresses. Selected pressure-sensitive proteins were partially sequenced and their genes were identified by reverse genetics. In a transcriptome analysis of a redundancy cleared shot gun library, about 7% of the genes investigated were found to be affected. Most of them appeared to be up-regulated 2- to 4-fold and these results were confirmed by real time PCR. Gene induction was shown for some genes up-regulated at the proteome level (clpL/groEL/rbsK), while the response of others to high hydrostatic pressure at the transcriptome level seemed to differ from that observed at the proteome level. The up-regulation of selected genes supports the view that the cell tries to compensate for pressure-induced impairment of translation and membrane transport.

High pressure-sensitive gene expression in Lactobacillus sanfranciscensis

Lactobacillus sanfranciscensis is a Gram-positive lactic acid bacterium used in food biotechnology. It is necessary to investigate many aspects of a model organism to elucidate mechanisms of stress response, to facilitate preparation, application and performance in food fermentation, to understand mechanisms of inactivation, and to identify novel tools for high pressure biotechnology. To investigate the mechanisms of the complex bacterial response to high pressure we have analyzed changes in the proteome and transcriptome by 2-D electrophoresis, and by microarrays and real time PCR, respectively. More than 16 proteins were found to be differentially expressed upon high pressure stress and were compared to those sensitive to other stresses. Except for one apparently high pressure-specific stress protein, no pressure-specific stress proteins were found, and the proteome response to pressure was found to differ from that induced by other stresses. Selected pressure-sensitive proteins were partially sequenced and their genes were identified by reverse genetics. In a transcriptome analysis of a redundancy cleared shot gun library, about 7 percent of the genes investigated were found to be affected. Most of them appeared to be up-regulated 2- to 4-fold and these results were confirmed by real time PCR. Gene induction was shown for some genes up-regulated at the proteome level (clpL/groEL/rbsK), while the response of others to high hydrostatic pressure at the transcriptome level seemed to differ from that observed at the proteome level. The up-regulation of selected genes supports the view that the cell tries to compensate for pressure-induced impairment of translation and membrane transport.

Barotolerance is inducible by preincubation under hydrostatic pressure, cold-, osmotic- and acid-stress conditions in Lactobacillus sanfranciscensis DSM 20451T.

AIMS: This study addresses the inducibility of barotolerance by preincubation of Lactobacillus sanfranciscensis DSM 20451T under various sublethal stress conditions. METHODS AND RESULTS: Stress conditions which reduce the growth rate of L. sanfranciscensis DSM 20451T to 10% of its maximum were determined. These conditions were met at 43, 12.5 degrees C, a pH value of 3.7, 1.9% NaCl, or 80 MPa respectively. In contrast to heat preincubation, other prestresses, including salt, cold and pressure led to an increase of barotolerance by hydrostatic pressure of 300 MPa for 30 min. Stationary-phase cells also showed an increased barotolerance. Sublethal pressure leads to enhanced heat tolerance. CONCLUSIONS: Stress response to salt, low temperature and acidic pH as well as starvation overlap with that one to high pressure by inducing barotolerance. SIGNIFICANCE AND IMPACT OF THE STUDY: Inactivation of bacteria by high pressure treatment is influenced by their history which modulates barotolerance. Mechanisms of barotolerance appear different from heat shock defence.

Characterization of lactobacilli towards their use as probiotic adjuncts in poultry.

AIMS: A total of 112 strains of lactic acid bacteria of duck origin were studied for their use as a probiotic feed supplement. METHODS AND RESULTS: In vitro studies included aggregation, co-aggregation, cell surface hydrophobicity and adhesion activities on poultry crop cells and human Hep2-cells. Additionally, growth with bile acids (chicken bile, ox gall and taurocholic acid) and tolerance to acidic pH were tested. Among all the isolates, two strains (Lactobacillus animalis TMW 1.972 and Lactobacillus salivarius TMW 1.992) were selected for a survival test in poultry. Monitoring and differentiation of these strains was achieved by selective detection as rifampicin and erythromycin double-resistant mutants. After a single feed administration, both micro-organisms were shown to persist in the crop and caecum of ducks for a period of 18 and 22 days, respectively. For identification of Lact. animalis and Lact. salivarius, two specific PCRs targeted against 16S rDNA were developed. CONCLUSIONS: Within the autochtoneous microflora of ducks, two strains of lactobacilli exhibited strong potential as probiotic adjuncts. The results indicate that the natural gut microflora of poultry serves as an excellent source for optimal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: A general strategy for the selection of probiotic strains is presented. The suggested sequence of tests allows identification of the most promising candidates within complex ecosystems or large strain collections with minimal expenditure.

Evidence for coupling of membrane targeting and function of the signal recognition particle (SRP) receptor FtsY.

Recent studies have indicated that FtsY, the signal recognition particle receptor of Escherichia coli, plays a central role in membrane protein biogenesis. For proper function, FtsY must be targeted to the membrane, but its membrane-targeting pathway is unknown. We investigated the relationship between targeting and function of FtsY in vivo, by separating its catalytic domain (NG) from its putative targeting domain (A) by three means: expression of split ftsY, insertion of various spacers between A and NG, and separation of A and NG by in vivo proteolysis. Proteolytic separation of A and NG does not abolish function, whereas separation by long linkers or expression of split ftsY is detrimental. We propose that proteolytic cleavage of FtsY occurs after completion of co-translational targeting and assembly of NG. In contrast, separation by other means may interrupt proper synchronization of co-translational targeting and membrane assembly of NG. The co-translational interaction of FtsY with the membrane was confirmed by in vitro experiments.

Cloning, expression, and characterization of acetate kinase from Lactobacillus sanfranciscensis.

In the metabolism of Lactobacillus sanfranciscensis, the acetate kinase (AK) is a key enzyme and responsible for dephosphorylation of acetyl phosphate with the concomitant production of acetate and ATP. The L. sanfranciscensis ack gene was identified by PCR methods. It encodes a 397 amino acid protein sharing 56% similarity with Bacillus subtilis AK. Whereas cotranscription of ack and pta (phosphotransacetylase) is reported in previously characterised organisms, the L. sanfranciscensis ack gene is not located in direct neighbourhood to the encoding gene. AK was heterologously expressed in E. coli and characterised by its v(max) and Km values and by the dependence of enzyme activity on temperature and pH. Based on this data the in vivo role of the enzyme is discussed.

A novel method to determine the topology of peroxisomal membrane proteins in vivo using the tobacco etch virus protease.

Most proteins essential for the biogenesis of peroxisomes (peroxins) that are identified to date are associated with or are integral components of the peroxisomal membrane. A prerequisite in elucidating their function is to determine their topology in the membrane. We have developed a novel tool to analyze the topology of peroxisomal membrane proteins in the yeast Hansenula polymorpha in vivo using the 27-kDa NIa protease subunit from the tobacco etch virus (TEVp). TEVp specifically cleaves peptides containing the consensus sequence, EXXYXQ downward arrowS (tev). We show that cytosolic TEVp and peroxisomal TEVp.SKL are selectively active on soluble cytosolic and peroxisomal tev-containing proteins in vivo, respectively, without affecting the viability of the yeast cells. The tev sequence was introduced in between the primary sequence of the peroxisomal membrane proteins Pex3p or Pex10p and the reporter protein enhanced green fluorescent protein (eGFP). Co-synthesis of these functional tev-GFP tagged proteins with either cytosolic TEVp or peroxisomal TEVp.SKL revealed that the C termini of Pex3p and Pex10p are exposed to the cytosol. Additional applications of the TEV protease to study peroxisome biogenesis are discussed.

In vitro stability and expression of green fluorescent protein under high pressure conditions.

AIMS: The objective of this work was to evaluate the use of wild-type GFP and mutant forms thereof as reporter for gene expression under high pressure conditions. METHODS AND RESULTS: The intensity of fluorescence after high pressure treatment was checked by subjecting cells, crude protein extracts containing GFPs and purified GFPs to pressures ranging from 100 MPa to 900 MPa. All tested GFP's retained fluorescence up to 600 MPa without loss of intensity. Expression of GFP under sublethal conditions was investigated in Escherichia coli with plasmid pQBI63, in which rsGFP is placed downstream of the T7 RNA polymerase binding site. T7 RNA polymerase is controlled in E. coli BL21 (DE3) pLysS by an IPTG inducible lacUV5 promoter. A pressure induced increase of GFP expression was monitored at 50 Mpa and 70 MPa. CONCLUSION: Fluorescence of GFPs is not influenced at pressures at which protein expression still occurs. We showed that the expression system used is inducible by pressurized conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proved GFP to be a suitable reporter for gene expression studies capable to detect pressure induced gene expression.

Characterization of transmembrane segments 3, 4, and 5 of MalF by mutational analysis.

MalF and MalG are the cytoplasmic membrane components of the binding protein-dependent ATP binding cassette maltose transporter in Escherichia coli. They are thought to form the transport channel and are thus of critical importance for the mechanism of transport. To study the contributions of individual transmembrane segments of MalF, we isolated 27 point mutations in membrane-spanning segments 3, 4, and 5. These data complement a previous study, which described the mutagenesis of membrane-spanning segments 6, 7, and 8. While most of the isolated mutations appear to cause assembly defects, L(323)Q in helix 5 could interfere more directly with substrate specificity. The phenotypes and locations of the mutations are consistent with a previously postulated structural model of MalF.

Cloning of the phosphotransacetylase gene from Lactobacillus sanfranciscensis and characterization of its gene product.

The phosphotransacetylase (PTA) (EC 2.3.1.8) catalyzes a key branch point reaction in the carbohydrate pathway of Lactobacillus sanfranciscensis. In this report, we describe the cloning of the pta gene. The DNA sequence analysis revealed a 987 bp open reading frame encoding a protein with a molecular mass of 35.5 kD. These are the first studies on a PTA of an organism representative for the heterofermentative lactic acid bacteria. Unlike in most other bacteria analysed so far, in L. sanfranciscensis the pta gene is not adjacent located to the gene encoding acetate-kinase. The PTA was heterologously expressed as a biotinylated fusion protein in E. coli and purified to homogeneity. Rate dependence on all substrates followed Michaelis-Menten kinetics. The apparent Km values for acetylphosphate and CoA (forward reaction) were 1.3 and 0.1 mM, respectively. The apparent Vmax was 194 U/mg. The enzyme also catalyzed in vitro the reverse reaction with apparent Km values for acetylCoA and phosphate of 0.6 and 6.7 mM, respectively (Vmax of 38 U/mg). The PTA showed a wide range of temperature for optimal activity (49 degrees C to 58 degrees C). It was inactivated after 15 min at 60 degrees C. Its activity was not affected by addition of MgCl2 (10 mM) or KCl (100 mM).