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At least 613 records · Page 34Linked to original sources
PubMed2005

Antimicrobial activity of conditioned medium fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni Hi5 insect cells.

Concentrated conditioned medium (CM) fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni cells, eluting from a gel filtration column at around 10 kDa, were found to exhibit strong antibacterial activity against Bacillus megaterium and Escherichia coli. The B. megaterium cells incubated in the CM fraction from Sf9 cells rapidly lost viability: after 8 min the viability had decreased to 0.7%, as compared with the control. Addition of the CM fraction to E. coli cells resulted in a less drastic drop in viability: 65% viability was lost after 60 min of incubation. Further, exposure to the CM fraction caused a substantial leakage of intracellular proteins, as demonstrated by SDS-PAGE analysis. Cell lysis was confirmed by optical density measurements, microscopic investigations and flow cytometry. B. megaterium exposed to a CM fraction from T. ni cells lost 97% of their viability in about 40 min. Ubiquitin, thioredoxin and cyclophilin were identified in the antibacterial fraction from Sf9 cells by mass spectrometry and N-terminal amino acid sequencing. Other proteins in the fraction gave no matches in a database search. Since ubiquitin was shown not to cause the antimicrobial effect and thioredoxin and cyclophilin were likely not involved, the responsible agent may be an unknown protein, not yet registered in databases. The antimicrobial effect of the CM fraction from T. ni cells most probably comes from a lysozyme precursor protein.

Animals↗
PubMed2006

A simple method for extracting C-phycocyanin from Spirulina platensis using Klebsiella pneumoniae.

C-phycocyanin (C-PC) was extracted from fresh Spirulina platensis by deploying a species of non-pathogenic nitrogen-fixing bacteria, namely, Klebsiella pneumoniae. The algal slurry was neither washed nor centrifuged; the bacterial culture was poured into the slurry, the vessel sealed, and crude C-PC extracted after about 24 h. The extraction was clean and efficient, and the purity and concentration of C-PC proved to be of adequate quality.

Bacterial Proteins↗
PubMed2006

Mutagenesis of a bacteriophage lytic enzyme PlyGBS significantly increases its antibacterial activity against group B streptococci.

Group B streptococci (GBS) are the leading cause of neonatal meningitis and sepsis worldwide. Intrapartum antibiotic prophylaxis (IAP) is the current prevention strategy given to pregnant women with confirmed vaginal GBS colonization. Due to antibiotic resistance identified in GBS, we previously developed another strategy using a bacteriophage lytic enzyme, PlyGBS, to reduce vaginal GBS colonization. In this study, various DNA mutagenesis methods were explored to produce PlyGBS mutants with increased lytic activity against GBS. Several hyperactive mutants were identified that contain only the endopeptidase domain found in the N-terminal region of PlyGBS and represent only about one-third of the wild-type PlyGBS in length. Significantly, these mutants not only have 18-28-fold increases in specific activities compared to PlyGBS, but they also have a similar activity spectrum against several streptococcal species. One of the hyperactive mutants, PlyGBS90-1, reduced the GBS colonization from >5 logs of growth per mouse to <50 colony-forming units (cfu) 4 h post treatment ( approximately 4-log reduction) using a single dose in a mouse vaginal model. A reduction in GBS colonization before delivery should significantly reduce neonatal GBS infection providing a safe alternative to IAP.

Animals↗
PubMed2000

Role of proteases in autolysis of Penicillium chrysogenum chemostat cultures in response to nutrient depletion.

An industrial strain of Penicillium chrysogenum was subjected to carbon or nitrogen limitation in a chemostat and the response monitored in terms of the "classical" indicators of autolysis (biomass decline and ammonia release), culture degradation (as measured by image analysis) and by obtaining profiles for three classes of proteases implicated in autolysis. Under both sets of conditions (carbon or nitrogen limitation), once started, autolysis involved a succession of different protease activities. The first stages of the process of autolysis in starved chemostat cultures was associated with peaks in the activities of both serine and aspartyl proteases, coinciding with the mobilisation of endogenous energy reserves. Conversely, a peak in the activity of metalloproteases was associated with the later stages of autolysis, perhaps occurring in response to depletion of endogenous energy reserves; the activity of these enzymes led to gross culture degradation, disintegration of ordered mycelial structures and signalled the end of metabolic activity (respiration) within the culture. These findings indicate that strategies intended to control/regulate autolysis in large-scale industrial fungal cultures might profitably be focused on regulation of the activity of key classes of proteases involved in the series of events leading to culture degradation.

Ammonia↗
PubMed1997

Improvement of culture conditions to overproduce beta-galactosidase from Escherichia coli in Bacillus subtilis.

The effect of some culture variables in the production of beta-galactosidase from Escherichia coli in Bacillus subtilis was evaluated. The lacZ gene was expressed in B. subtilis using the regulatory region of the subtilisin gene aprE. The host contained also the hpr2 and degU32 mutations, which are known to overexpress the aprE gene. We found that, when this overproducing B. subtilis strain was grown in mineral medium supplemented with glucose (MMG), beta-galactosidase production was partially growth-associated, as 40%-60% of the maximum enzyme activity was produced before the onset of the stationary phase. In contrast, when a complex medium was used, beta-galactosidase was produced only at low levels during vegetative growth, whereas it accumulated to high levels during early stationary phase. Compared with the results obtained in complex media, a 20% increase in specific beta-galactosidase activity in MMG supplemented with 11.6 g/l glucose was obtained. On the 1-1 fermenter scale, a three-fold increase in volumetric beta-galactosidase activity was obtained when the glucose concentration was varied from 11 g/l to 26 g/l. In addition, glucose feeding during the stationary phase resulted in a twofold increase in volumetric enzyme activity as cellular lysis was prevented. Finally, we showed that oxygen uptake and carbon dioxide evolution rates can be used for on-line determination of the onset of stationary phase, glucose depletion and biomass concentration.

Bacillus subtilis↗
PubMed1999

Autolysis of Escherichia coli and Bacillus subtilis cells in low gravity.

The role of gravity in the autolysis of Bacillus subtilis and Escherichia coli was studied by growing cells on Earth and in microgravity on Space Station Mir. Autolysis analysis was completed by examining the death phase or exponential decay of cells for approximately 4 months following the stationary phase. Consistent with published findings, the stationary-phase cell population was 170% and 90% higher in flight B. subtilis and E. coli cultures, respectively, than in ground cultures. Although both flight autolysis curves began at higher cell densities than control curves, the rate of autolysis in flight cultures was identical to that of their respective ground control rates.

Bacillus subtilis↗
PubMed2002

Modification of the peptidoglycan of Escherichia coli in the viable but nonculturable state.

The aim of this study was to analyse the chemical composition of peptidoglycan and the state of some of the enzymes involved in its metabolism in Escherichia coli KN126 in the viable but nonculturable (VBNC) state which is a survival strategy adopted by bacteria (including those of medical interest) when exposed to environmental stresses. When entering the VBNC state, E. coli cells miniaturised and became coccus-shaped. Analysis of peptidoglycan chemical composition, by separation in HPLC of muropeptides released by muramidase digestion of purified peptidoglycan, indicated a high degree of cross-linking, a threefold increase in unusual DAP-DAP cross-linking, an increase in muropeptides bearing covalently bound lipoprotein, and a shortening of the average length of glycan strands in comparison with dividing cells. Analysis of penicillin-binding proteins (PBPs), enzymes involved in the terminal stage of peptidoglycan assembly showed the disappearance of high-molecular-weight PBPs 1A, 1B, 2, and 3 in VBNC cells. Finally, VBNC cells displayed an autolytic capability which was far higher than that of exponentially growing cells. It is suggested that part of these alterations of peptidoglycan may be connected with the VBNC state.

Antigens, Bacterial↗
PubMed2004

Histone H1 as a reporter protein to investigate glycation in bacteria.

Nonenzymatic glycosylation (glycation) of proteins is a multistage chemical process starting as a condensation reaction between reducing sugars and primary amino groups (mainly from the side chains of Lis and Arg) and ending up with formation of complex heterocyclic compounds called advanced glycation end products (AGEs). For a long time, glycation has been attributed to the long-lived eukaryotes (including in humans) only. In a recent study, we showed that glycation also occurs in bacteria. The present study aims to prove that bacterial cytoplasm contains soluble glycating compounds. To this end, Lis/Arg-rich histone H1 isolated from rat liver was treated with deproteinized Escherichia coli cytoplasm through a dialysis membrane. This treatment leads to accumulation of AGEs as well as to a remarkable degradation of the reporter protein on storage at 4 degrees C. Our results indicate also that glycation can be inhibited by acetylsalicylic acid (aspirin), thiamine (vitamin B1), and pyridoxine (vitamin B6).

Animals↗
PubMed2005

Isolation and characterization of a virulent Lactobacillus sanfranciscensis bacteriophage and its impact on microbial population in sourdough.

Thirty-five sourdough samples used for sweet and salted Italian baked products were checked for the presence of a virus active on Lactobacillus sanfranciscensis species. One phage, named EV3, was isolated and its phenotypic and genotypic features were investigated. It belonged to the Siphoviridae family (morphotype B1); its life cycle at 25 degrees C lasted 3 h with a burst size of about 30 viral particles per infected cell. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed one major structural protein of 35 kDa and four minor proteins. The genome, approximately 32 kb long, was a double-stranded linear DNA molecule with a pac-type system. Phage spreading into sourdough did not adversely affect acidification and volume increase of the dough neither lactobacilli counts; the propagation of viral particles was shown to be hindered. This is the first report of the isolation of a L. sanfranciscensis phage.

Bacteriolysis↗
PubMed2005

Thermophilin 110: a bacteriocin of Streptococcus thermophilus ST110.

A screen of thermophilic lactic acid bacteria identified Streptococcus thermophilus strain ST110 as the putative producer of a bacteriocin with high level of activity against pediococci. Thermophilin 110 was isolated from culture supernatant after 16 h of growth and partially purified by a chloroform extraction procedure. The bacteriocin inhibited the growth of several lactic acid bacteria and in the case of Pediococcus acidilactici, it induced cell lysis with the concomitant release of OD260-absorbing material and intracellular enzymes. SDS-PAGE analysis revealed two components with estimated sizes between 4.0 kDa and 4.5 kDa, respectively, with possible involvement in bacteriocin activity as indicated by agar overlay assays with P. acidilactici as the target organism. Thermophilin 110 was inactivated by several proteolytic enzymes and also by alpha-amylase, which indicated the putative requirement of a glycosidic component for activity. The bacteriocin produced by S. thermophilus may be especially useful in the food processing industries to control spoilage caused by pediococci.

Anti-Bacterial Agents↗
PubMed2000

Formation and stability of the bacteriophage lambda replication complexes in UV-irradiated Escherichia coli.

Bacteriophage lambda replication complex, containing the phage-encoded O initiator protein protected from proteases by other elements of this complex, is a stable structure that can be inherited by one of the two daughter lambda DNA copies after a replication round in Escherichia coli. In normal growth conditions in bacteria bearing a plasmid derived from bacteriophage lambda, such a complex may be stable for many cell generations. However, it was found that this stable structure is disassembled under certain conditions, namely, after heat shock. Therefore, we asked whether other environmental stresses may cause disassembly of the lambda replication complex. We found that UV irradiation of the host cells prevented formation of the stable lambda replication complex (though not preventing phage replication), while the same UV doses did not affect the stability of the replication complex assembled prior to the irradiation. These results indicate that the stable lambda replication complex, although sensitive to heat shock, is resistant to some other environmental stresses and that formation of at least two types of lambda replication complexes is possible. Both stable and unstable lambda replication complexes are functional because replication of lambda DNA under conditions preventing formation of the stable complex proceeds efficiently.

Bacteriolysis↗
PubMed2001

Lactobacillus acidophilus autolysins inhibit Helicobacter pylori in vitro.

Antibacterial activity of 17 strains of lactobacilli was tested against 10 strains of H. pylori. The inhibition observed was related to the acid production and the low pH attained. No relationship between CagA phenotype of H. pylori strains and tolerance to lactic acid was observed. In mixed cultures, L. acidophilus CRL 639 showed an autolytic behavior after 24 h of culture. At this moment, H. pylori CCUG17874 showed a decrease of 2 log-cycle, and no viable count was detected after 48 h. The bactericidal effect of L. acidophilus CRL 639 in mixed cultures is related to a proteinaceous compound released after cell lysis.

Antibiosis↗
PubMed1997

The ability of 2-deoxyglucose to promote the lysis of Streptococcus bovis JB1 via a mechanism involving cell wall stability.

The non-metabolizable glucose analog, 2-deoxyglucose (2-DG), decreased the growth rate and optical density of Streptococcus bovis JB1 20%, but it had an even greater effect on stationary phase cultures. Control cultures receiving only glucose (2 mg/ml) lysed very slowly (<5% decline in optical density in 48 h), but cultures that had been grown with glucose and 2-DG (2 mg/ml each) lysed much faster (>85% decline in optical density in 48 h). Cultures that were treated with inhibitors that decreased intracellular ATP (sodium fluoride, nigericin, and valinomycin or tetrachlorosalicylanilide) or membrane potential (sodium fluoride, nigericin, and valinomycin, tetrachlorosalicylanilide, or phenylmethylsulfonyl fluoride) did not promote lysis. 2-DG had its greatest effect when it was added at inoculation. If 2-DG was added at later times, less lysis was observed, and cells that were given 2-DG just prior to stationary phase were unaffected. Cells that were grown with glucose and 2-DG were more susceptible to cell wall-degrading enzymes (lysozyme and mutanolysin) than cells that had been grown only with glucose, but sublethal doses of penicillin during growth did not promote lysis after the cells had reached stationary phase. The idea that 2-DG might be affecting autolytic activity was supported by the observation that cultures washed and resuspended in fresh medium with or without 2-DG lysed at a slower rate than cultures that were not centrifuged or were resuspended in the culture supernatant.

Adenosine Triphosphate↗
PubMed2001

Bacteriophage P4282, a parasite of Ralstonia solanacearum, encodes a bacteriolytic protein important for lytic infection of its host.

To enhance bacterial wilt resistance in tobacco expressing a foreign protein, we isolated the bacteriolytic gene from a bacteriophage that infects Ralstonia solanacearum. The bacteriolytic protein of phage P4282 isolated in Tochigi Prefecture was purified from a lysate of R. solanacearum M4S cells infected with the phage, and its bacteriolytic activity was assayed by following the decrease in the turbidity of suspensions of R. solancacearum M4S cells. The molecular weight of the bacteriolytic protein was approximately 71 kDa, and the sequence of the N-terminal 13 amino acids was determined. We used oligonucleotide probes based on this amino acid sequence to isolate the bacteriolytic gene from phage P4282 DNA. This gene of 2061 bp encodes a product of 687 amino acids, whose calaculated molecular weight was 70.12 kDa. The bacteriolytic gene was placed under the control of an inducible promoter. and the plasmid was transformed into Escherichia coli NM522. The soluble proteins extracted from E.coli NM522 cells harboring the plasmid with the bacteriolytic gene showed obvious bacteriolytic activities against several strains of R. solanacearum isolated in various districts in Japan. DNA fragments from five phages, isolated in Niigata, Aomori, Okinawa, Fukushima and Yamaguchi Prefectures, hybridized to the bacteriolytic gene of phage P4282. These observations indicate that the bacteriolytic protein shows nonspecific activity against R. solanacearum strains, and a sequence similar to that of the bacteriolytic gene is conserved in the DNA of other bacteriophages. These results indicate that the generation of transgenic (tobacco) plants expressing the bacteriolytic gene of phage P4282 might result in enhanced resistance to bacterial wilt in tobacco.

Bacteriolysis↗
PubMed2001

Analysis of a regulator involved in the genetic switch between lysis and lysogeny of the temperate Lactococcus lactis phage phi LC3.

Sequencing of a 1.3-kb fragment of DNA from the temperate Lactococceus lactis subsp. cremoris phage phiLC3 revealed a pair of two divergently oriented ORFs, orf63 and orf286. The deduced amino acid sequence of the product of orf286 showed extensive homology to those of repressors of the temperate lactococcal phages rlt, Tuc2009 and BK5-T. A mutant with an amber mutation in orf286 gave rise to a clear plaque phenotype, indicating that this gene is involved in the lytic and lysogenic development of phiLC3. Gel mobility shift assays showed that the partially purified Orf286 protein bound specifically to the 224-bp intergenic region located between orf286 and orf63, and further characterization by DNase I footprinting analysis revealed that Orf286 protects two distinct sites within this region. Sequence analysis of the intergenic region revealed two putative, divergently oriented promoters, P1 and P2; orf286 and orf63 are probably transcribed from P1 and P2, respectively. In vivo analyses of P1 and P2 using beta-galactosidase as a reporter enzyme in L. lactis showed that transcription from P1 was repressed while transcription from P2 was stimulated in the presence of the Orf286 protein. These results suggest a complex role for the Orf286 protein in regulating the genetic switch between lytic and lysogenic growth of phiLC3.

Bacteriolysis↗
PubMed2001

Rapid degradation of bacteriophage lambda O protein by ClpP/ClpX protease influences the lysis-versus-lysogenization decision of the phage under certain growth conditions of the host cells.

The initiator of bacteriophage lambda DNA replication, the O protein, is rapidly degraded in Escherichia coli by the ClpP/ClpX protease encoded by the host. Although the biochemical mechanism of this degradation has been investigated intensively, a physiological role for this process remained unknown since little effect of dysfunction of clpP and clpX genes on the lytic development of the phage was observed. Here we demonstrate that activities of clpP and clpX genes influence the lysis-versus-lysogenization decision of bacteriophage lambda under certain growth conditions of the host cells. This decision is influenced specifically by ClpP/ClpX-mediated O degradation and resultant inhibition of early lambda DNA replication because mutations in clpP and clpX genes have little effect on stability of other lambda proteins involved in the regulation of the phage developmental switch.

Adenosine Triphosphatases↗
PubMed2006

Permeabilization and lysis induced by bacteriocins and its effect on aldehyde formation by Lactococcus lactis.

Permeabilization induced by lacticin 3147, lactococcins A, B and M, enterocin AS-48 and nisin, bacteriocins described as cell membrane-pore forming and lytic agents, enhanced in all cases aldehyde formation by Lactococcus lactis IFPL730. Nevertheless, the conversion of isoleucine into 2-methylbutyraldehyde depended not only on the degree of permeabilization but also on the bacteriocin that caused the cell membrane damage. The highest values of 2-methylbutyraldehyde corresponded to cell suspensions containing lacticin 3147 and lactococcins, treatments that provoked further lysis in addition to induced permeabilization.

Aldehydes↗