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Showing papers in "Microbiology in 1992"


Journal ArticleDOI
TL;DR: A physiological link is proposed between sucrose fermentation capacity and nisin production ability and carbon source regulation appears to be a major control mechanism for nisinProduction.
Abstract: Summary: Nisin production by Lactococcus lactis subsp. lactis NIZO 22186 was studied in batch fermentation using a complex medium. Nisin production showed primary metabolite kinetics: nisin biosynthesis took place during the active growth phase and completely stopped when cells entered the stationary phase. A stringent correlation could be observed between the expression of the prenisin gene (nisA) and the synthesis of the post-translationally enzymically modified and processed mature nisin peptide. Moreover, it seemed likely that nisin had a growth control function. A physiological link is proposed between sucrose fermentation capacity and nisin production ability. Carbon source regulation appears to be a major control mechanism for nisin production.

276 citations


Journal ArticleDOI
TL;DR: Twenty-five strains of lactobacilli were tested for their ability to adhere to human enterocyte-like Caco-2 cells in culture and a high level of calcium-independent adhesion was observed with the human stool isolate Lactobacillus acidophilus strain LB.
Abstract: Twenty-five strains of lactobacilli were tested for their ability to adhere to human enterocyte-like Caco-2 cells in culture. Seven Lactobacillus strains adhered well to the Caco-2 cells, of which three possessed calcium-independent adhesion properties. A high level of calcium-independent adhesion was observed with the human stool isolate Lactobacillus acidophilus strain LB. Scanning electron microscopy revealed that this strain adhered to the apical brush border of the cells. Adhesion increased in parallel with the morphological and functional differentiation of the Caco-2 cells. Two Lactobacillus components were involved in this adhesion. One was protease-resistant and bacterial-surface-associated; the other was heat-stable, extracellular and protease-sensitive.

263 citations


Journal ArticleDOI
TL;DR: Microsequencing of the pure bacteriocin and of tryptic fragments showed that mesentericin Y105 is a 36 amino acid polypeptide whose primary structure is close to that of leucocin A-UAL 187, which contains an extra residue at the C-terminus and displays only two differences in the overlapping sequence.
Abstract: SUMMARY: A Leuconostoc mesenteroides ssp. mesenteroides was isolated from goat's milk on the basis of its ability to inhibit the growth of Listeria monocytogenes. The antimicrobial effect was due to the presence in the culture medium of a compound, named mesentericin Y105, excreted by the Leuconostoc mesenteroides Y105. The compound displayed known features of bacteriocins from lactic acid bacteria. It appeared as a proteinaceous molecule exhibiting a narrow inhibitory spectrum limited to genus Listeria. The apparent relative molecular mass, as indicated by activity detection after SDS-PAGE, was 2.5–3.0 kDa. The bacteriocin was purified to homogeneity by a simple three-step procedure: a crude supernatant obtained from an early-stationary-phase culture in a defined medium was subjected to affinity chromatography on a blue agarose column, followed by ultrafiltration through a 5 kDa cut-off membrane, and finally by reverse-phase HPLC on a C4 column. Microsequencing of the pure bacteriocin and of tryptic fragments showed that mesentericin Y105 is a 36 amino acid polypeptide whose primary structure is close to that of leucocin A-UAL 187, which contains an extra residue at the C-terminus and displays only two differences in the overlapping sequence. However, unlike leucocin A-UAL 187, mesentericin Y105 displayed a bactericidal mode of action.

246 citations


Journal ArticleDOI
TL;DR: The regulatory unit of Bacillus subtilis strain 168 encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and of its modifier has been sequenced, and found to be a divergon consisting of divergently transcribed operons lytABC and lytR.
Abstract: The regulatory unit of Bacillus subtilis strain 168 encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and of its modifier has been sequenced, and found to be a divergon consisting of divergently transcribed operons lytABC and lytR. Proteins LytA, LytB and LytC are endowed with export signal peptides. Mature LytA is a 9.4 kDa, highly acidic polypeptide whose deduced amino acid sequence points to a lipoprotein. LytB and LytC, the modifier and the amidase, are highly basic. After cleavage of the signal sequence their molecular masses are 74.1 and 49.9 kDa, respectively. These two proteins share considerable homology in their N-terminal moieties and have three GSNRY consensus motifs, characteristic of nearly all amidases. The C-terminal moiety of LytB exhibits homology to the product of spollD. LytR is a 35 kDa protein which acts as an attenuator of the expression of both lytABC and lytR operons. Transcription of the lytABC operon proceeds from two promoters: PD, identified as P28–7 (Gilman et al., 1984), and an upstream PA. The former only is subject to LytR attenuation. Translational initiation of lytB and lytC is directed by UUG start codons, suggesting that lytA, B and C undergo coupled translation. Transcription of lytR is initiated at two start sites, one of which corresponds to a highly intense PA promoter whereas the other does not seem to share much homology with any of the known promoter consensus sequences. Both promoters are attenuated by LytR. It is confirmed that the synthesis of the amidase is controlled at least in part by SigD, i.e. that it belongs to the fla regulon and that its activity, or part of it, is co-regulated with flagellar motility. The role of the mutations conferring the Sin, Fla and Ifm phenotypes in the expression of the lytABC operon is discussed.

241 citations


Journal ArticleDOI
TL;DR: RRNA sequences were used as targets for synthetic oligonucleotides labelled with the fluorescent dye tetramethylrhodamine isothiocyanate (Tritc) for in situ hybridizations to detect micro-organisms directly in soils that have different contents of soil minerals and organic material.
Abstract: SUMMARY: rRNA sequences were used as targets for synthetic oligonucleotides labelled with the fluorescent dye tetramethylrhodamine isothiocyanate (Tritc) for in situ hybridizations to detect micro-organisms directly in soils that have different contents of soil minerals and organic material. Introduced Pseudomonas aeruginosa cells were directly fixed in soils and applied to slides after separation of large soil minerals only. Remaining soil minerals (clay minerals) and organic material (up to 8%) did not significantly interfere with signal expression after hybridization. Background signals were mainly caused by autofluorescence of organic material. Non-specific binding of labelled oligonucleotides to soil particles was not observed. In situ detection of introduced cells of Pseudomonas cepacia in a sandy loam spiked with a mixture of selected soil micro-organisms was possible after hybridization with a specific probe. Analysis of natural bacterial populations in soil, however, was not possible by in situ hybridization without activation of these micro-organisms by adding nutrients. Growing cells, e.g. Streptomyces scabies hyphae growing in amended soil, were easily detected.

228 citations


Journal ArticleDOI
TL;DR: The phenomenon of bacterial bioluminescence can now be captured and applied within any bacterial species from several rather different perspectives and provides a real-time noninvasive reporter for measuring gene expression, a sensitive marker for bacterial detection and a measure of intracellular biochemical function, i.e. as a holistic determinant of cellular viability.
Abstract: Luminous bacteria constitute some of the most fascinating subjects in microbiology and are much more prevalent than is frequently appreciated. They are found in marine, freshwater and terrestrial environments and can be most easily isolated as saprophytes growing on dead fish or meat. Fig. 1 (a) shows the bioluminescence emission from a fresh fish-market sprat imaged using a photon video camera. This light, which can be obtained from almost any fish, is derived from bioluminescent bacteria. The diversity of these micro-organisms is such that only a very small percentage have been studied biochemically and even fewer have been the subject of genetic analysis. There may, therefore, be much yet to discover about the diverse biochemistry and genetics of bacterial bioluminescence. Nevertheless, through the techniques of genetic engineering, the phenomenon of bacterial bioluminescence can now be captured and applied within any bacterial species from several rather different perspectives. It provides a real-time noninvasive reporter for measuring gene expression, a sensitive marker for bacterial detection and a measure of intracellular biochemical function, i.e. as a holistic determinant of cellular viability. This review aims to provide the necessary background to examine recent developments in these areas and thereby encourage a growing awareness for the multi-faceted nature of in vivo bioluminescence.

215 citations


Journal ArticleDOI
TL;DR: Exponential phase cells of the yeast when treated with a non-lethal concentration of hydrogen peroxide for 60 min adapted to become resistant to the lethal effects of a higher dose of H2O2, indicating that the adaptive response does not require functional mitochondria.
Abstract: SUMMARY: Exponential phase cells of the yeast Saccharomyces cerevisiae when treated with a non-lethal concentration of hydrogen peroxide (H2O2; 0·2 mM) for 60 min adapted to become resistant to the lethal effects of a higher dose of H2O2 (2 mM). From studies using cycloheximide to inhibit protein synthesis it appears that protein synthesis is required for maximal induction of resistance but that some degree of protection from the lethal effects of peroxide can be acquired in the absence of protein synthesis. Treatment of cells with 50 μg cycloheximide ml-1 alone led to them acquirng some protection from peroxide. Cells subjected to heat shock became more resistant to 2 mM-H2O2; however, peroxide pretreatment did not confer thermotolerance. L-[35S]Methionine labelling of cells subjected to 0·2 mM-H2O2 stress showed that synthesis of at least ten polypeptides was induced by peroxide treatment. Some of these were also induced in cells subjected to heat shock (23 to 37 °C shift) but the synthesis of at least four polypeptides (45,39·5,38 and 24 kDa) was unique to peroxide-stressed cells. Resistance to peroxide was also inducible in an isogenic petite and an isogenic strain with a mutation in the HAP1 gene, indicating that the adaptive response does not require functional mitochondria.

214 citations


Journal ArticleDOI
TL;DR: Sakacin A, a bacteriocin produced by Lactobacillus sake Lb706 and which inhibits the growth of Listeria monocytogenes, was purified to homogeneity by ammonium sulphate precipitation and ion-exchange, hydrophobic-interaction and reversed-phase chromatography.
Abstract: SUMMARY: Sakacin A, a bacteriocin produced by Lactobacillus sake Lb706 and which inhibits the growth of Listeria monocytogenes, was purified to homogeneity by ammonium sulphate precipitation and ion-exchange, hydrophobic-interaction and reversed-phase chromatography. The complete amino acid sequence of sakacin A was determined by Edman degradation. The bacteriocin consisted of 41 amino acid residues and had a calculated M r of 4308.7, which is in good agreement with the value determined by mass spectrometry. The structural gene encoding sakacin A (sakA) was cloned and sequenced. The gene encoded a primary translation product of 59 amino acid residues which was cleaved between amino acids 18 and 19 to yield the active sakacin A. Sakacin A shared some sequence similarities with other bacteriocins.

209 citations


Journal ArticleDOI
TL;DR: The most predominant solutes detected within a wide range of marine and halophilic micro-organisms were two recently discovered tetrahydropyrimidines ectoine and hydroxyectoine, which were synthesized in response to osmotic stress.
Abstract: The aim of this investigation was to perform an extensive screening using HPLC and 13C-NMR spectroscopy to disclose the spectrum of osmolytes produced by aerobic heterotrophic and anoxygenic phototrophic eubacteria. The most predominant solutes detected within a wide range of marine and halophilic micro-organisms were two recently discovered tetrahydropyrimidines ectoine and hydroxyectoine, which were synthesized in response to osmotic stress.

207 citations


Journal ArticleDOI
TL;DR: This review concerns the mechanisms responsible for this stability and focusses on the active partition systems which may provide insights into mitotic processes in bacteria, if they exist.
Abstract: Bacterial plasmids, by definition, are not essential for host cell survival except in specialized environments where plasmid-borne genes, like those for antibiotic resistance or degradative metabolic pathways, confer a selective advantage. Although some plasmids are lost quite rapidly when appropriate selective pressure is removed, others are found to be retained over many generations in the absence of any selection. This implies that many plasmids do not rely for their stability on constant selection for the genes they carry. This review concerns the mechanisms responsible for this stability and focusses on the active partition systems which may provide insights into mitotic processes in bacteria, if they exist (see Austin, 1988; Nordstrom & Austin, 1989, for previous reviews). The inheritance of a plasmid on the basis of random segregation alone can be expressed by the relationship

200 citations


Journal ArticleDOI
TL;DR: A bacteriocin produced by Pediococcus acidilactici has been purified to homogeneity by a rapid and simple four-step purification procedure which includes ammonium sulphate precipitation, chromatography with a cation-exchanger and Octyl Sepharose, and reverse-phase chromatography, indicating an approximately 80,000-fold increase in the specific activity and about a 6-fold rise in the total activity.
Abstract: A bacteriocin produced by Pediococcus acidilactici has been purified to homogeneity by a rapid and simple four-step purification procedure which includes ammonium sulphate precipitation, chromatography with a cation-exchanger and Octyl Sepharose, and reverse-phase chromatography. The purification resulted in an approximately 80000-fold increase in the specific activity and about a 6-fold increase in the total activity. The amino acid composition and sequencing data indicated that the bacteriocin contained 43–44 amino acid residues. The predicted M r and isoelectric point of the bacteriocin are about 4600 and 8.6, respectively. Comparing the amino acid sequence of this bacteriocin with the sequences of leucocin A-UAL 187, sakacin P and curvacin A (bacteriocins produced by Leuconostoc gelidum, Lactobacillus sake and Lactobacillus curvatus, respectively) revealed that all four bacteriocins had in their N-terminal region the sequence Tyr-Gly-Asn-Gly-Val-Xaa-Cys, indicating that this concensus sequence is of fundamental importance for this group of bacteriocins. The bacteriocin from P. acidilactici and sakacin P were very similar, having at least 25 common amino acid residues. The sequence similarity was greatest in the N-terminal half of the molecules – 17 of the first 19 residues were common – indicating the fundamental importance of this region. Leucocin A-UAL 187 and curvacin A had, respectively, at least 16 and 13 amino acid residues in common with the bacteriocin from P. acidilactici.

Journal ArticleDOI
TL;DR: The polymerase chain reaction was used to amplify small-subunit ribosomal DNA from the anaerobic ciliated protozoon Metopus palaeformis, and from its uncultured endosymbiotic bacteria, but the present data were insufficient to resolve the detailed relationship between Blepharisma and Metopus and thus to determine if the heterotrichs are mono- or paraphyletic.
Abstract: The polymerase chain reaction (PCR) was used to amplify small-subunit ribosomal DNA from the anaerobic ciliated protozoon Metopus palaeformis, and from its uncultured endosymbiotic bacteria. This was accomplished directly from total DNA extracted from protozoa without prior isolation or enrichment for symbiont cells. The double-stranded amplification products were precipitated and directly sequenced using the linear PCR reaction. Fluorescent oligonucleotide probes were designed and used in whole-cell hybridizations to provide direct visual evidence that the sequences originated from the host ciliate and from the endosymbiont. Phylogenetic analysis of the Metopus palaeformis sequence consistently placed it as a deep-branching lineage near the root of the ciliate tree. However, the present data were insufficient to resolve the detailed relationship between Blepharisma and Metopus and thus to determine if the heterotrichs are mono- or paraphyletic. Phylogenetic analysis of the symbiont partial sequence clearly demonstrated that it is an archaeobacterium and that it is closely related to, but distinct from, Methanobacterium formicicum.

Journal ArticleDOI
TL;DR: The pretreatment of cells with low salt, however, was less effective in the induction of thermotolerance than a preceding mild heat stress and a mild heat shock induced a cross-protection against lethal salt stress.
Abstract: Bacillus subtilis induced a set of general stress proteins in response to a salt or heat stress. Cells subjected to a mild heat stress showed a protective response which enabled them to survive otherwise lethal temperatures (e.g. 52 °C). In a similar way bacteria were enabled to survive toxic concentrations of NaCl by pretreatment with lower salt concentrations. A mild heat shock induced a cross-protection against lethal salt stress. The pretreatment of cells with low salt, however, was less effective in the induction of thermotolerance than a preceding mild heat stress. Three stress proteins were identified on the basis of their N-terminal amino acid sequences as homologues of GroEL, DnaK and ClpP of Escherichia coli. The role of general and specific stress proteins in the induction of thermotolerance/salt tolerance and cross-protection is discussed.

Journal ArticleDOI
TL;DR: Freeze-etched preparations of intact cells have demonstrated that this protein layer is an oblique surface layer (S-layer) lattice which completely covers the cell surface.
Abstract: Summary: In a previous study, electron microscopic examinations of thin sections of Lactobacillus helveticus ATCC 12046 revealed a three-layered structure of the cell wall. The outermost component was identified as a layer of a non-glycosylated 52 kDa protein. Freeze-etched preparations of intact cells have now demonstrated that this protein layer is an oblique surface layer (S-layer) lattice (a = 4·5 nm, b = 9·6 nm, γ = 77 °) which completely covers the cell surface. Treatment with 5 m-LiCl extracted the S-layer protein from intact cells efficiently and selectively. Viability did not decrease significantly. Moreover, the S-layer reappeared when treated cells were allowed to grow again. In vitro self-assembly products obtained upon aggregation of isolated S-layer subunits exhibited the same oblique S-layer symmetry as observed on intact cells in vivo. The purified S-layer protein had a high content (44%) of hydrophobic amino acids. The N-terminal sequence was mainly composed of alanine, threonine, asparagine and aspartic acid.

Journal ArticleDOI
TL;DR: A survey of colicins in the ECOR reference collection of Escherichia coli is presented, and it is suggested that ColE1 plasmids comprise a stable (cohesive) plasmid lineage, while ColIa Plasmids represent a family of distinct plasmID lineages united by the presence of the colicin Ia operon.
Abstract: A survey of colicins in the ECOR reference collection of Escherichia coli is presented. Twenty-five of the 72 ECOR strains exhibited a phenotype consistent with colicin production and E. coli isolated from human hosts were more likely to be colicinogenic than those from animal hosts. Multiple representatives of two Col plasmids, lowmolecular-mass ColE1 plasmids and high-molecular-mass, conjugative ColIa plasmids were isolated from the ECOR collection and were examined with a combination of restriction fragment and Southern analysis. These data suggested that ColE1 plasmids comprise a stable (cohesive) plasmid lineage, while ColIa plasmids represent a family of distinct plasmid lineages united by the presence of the colicin Ia operon.

Journal ArticleDOI
TL;DR: The results indicate that, in addition to chitinase, lysis requires other gene products that may become limiting, such as demethylallosamidin, a much better protector against lysis.
Abstract: Summary: Previous results [E. Cabib, A. Sburlati, B. Bowers & S. J. Silverman (1989) Journal of Cell Biology 108, 1665-1672] strongly suggested that the lysis observed in daughter cells of Saccharomyces cerevisiae defective in chitin synthase 1 (Chs1) was caused by a chitinase that partially degrades the chitin septum in the process of cell separation. Consequently, it was proposed that in wild-type cells, Chs1 acts as a repair enzyme by replenishing chitin during cytokinesis. The chitinase requirement for lysis has been confirmed in two different ways: (a) demethylallosamidin, a more powerful chitinase inhibitor than the previously used allosamidin, is also a much better protector against lysis and (b) disruption of the chitinase gene in chs1 cells eliminates lysis. Reintroduction of a normal chitinase gene, by transformation of those cells with a suitable plasmid, restores lysis. The percentage of lysed cells in strains lacking Chs1 was not increased by elevating the chitinase level with high-copy-number plasmids carrying the hydrolase gene. Furthermore, the degree of lysis varied in different chs1 strains; lysis was abolished in chs1 mutants containing the scs1 suppressor. These results indicate that, in addition to chitinase, lysis requires other gene products that may become limiting.

Journal ArticleDOI
TL;DR: Lactobacillus plantarum strain 4B2, which exhibits a strong autoaggregating phenotype, receives the broad-host-range plasmid pAMβ1 with conjugation efficiencies as high as 10-2 transconjugants per donor using solid matings; broth matings also occur, but at low transfer frequencies.
Abstract: Summary: Lactobacillus plantarum strain 4B2, which exhibits a strong autoaggregating phenotype, receives the broad-host-range plasmid pAMβ1 with conjugation efficiencies as high as 10-2 transconjugants per donor using solid matings; broth matings also occur, but at low transfer frequencies. Filter-sterilized spent supernatant of this strain contains a 32 kDa protein that promotes aggregation, and consequently a high frequency of conjugation, in lactic acid bacteria containing α-1,2-glucose-substituted lipoteichoic or teichoic acids. It appears, therefore, that the substituted lipoteichoic or teichoic acids act as receptors for the aggregation-promoting protein.

Journal ArticleDOI
TL;DR: An insertion of about 100 bases within the central part of the 23S rRNA genes was found to be a phylogenetic marker for the bacterial line of descent of Gram-positive bacteria with a high DNA G + C content.
Abstract: An insertion of about 100 bases within the central part of the 23S rRNA genes was found to be a phylogenetic marker for the bacterial line of descent of Gram-positive bacteria with a high DNA G+C content. The insertion was present in 23S rRNA genes of 64 strains representing the major phylogenetic groups of Gram-positive bacteria with a high DNA G+C content, whereas it was not found in 23S rRNA genes of 55 (eu)bacteria representing Gram-positive bacteria with a low DNA G+C content and all other known (eu)bacterial phyla. The presence of the insertion could be easily demonstrated by comparative gel electrophoretic analysis of in vitro-amplified 23S rDNA fragments, which contained the insertion. The nucleotide sequences of the amplified fragments were determined and sequence similarities of at least 44% were found. The overall similarity values are lower than those of 16S and 23S rRNA sequences of the particular organism. Northern hybridization experiments indicated the presence of the insertion within the mature 23S rRNA of Corynebacterium glutamicum.

Journal ArticleDOI
TL;DR: In an attempt to focus attention on the desirability of developing an effective vaccine to protect against pneumococcal infection, this situation was likened, in terms of incidence and mortality, to that of poliomyelitis prior to the introduction of the vaccine.
Abstract: Streptococcus pneumoniae is an important agent of disease in man at the extremes of age and in individuals with underlying debilitating conditions. It is responsible for the majority of cases of community-acquired pneumonia, an important cause of septicaemia, one of the three most common pathogens in bacterial meningitis and the most prevalent agent in otitis media (Roberts, 1985). The precise incidence of each of these infections is not known because of problems in diagnosis (particularly with respect to pneumonia and otitis media) and inadequate reporting of cases. However, Austrian (19826), having reviewed a number of studies, concluded that in the USA there are 2-5 cases of pneumonia per 1000 population per annum, equating to between 440000 and 1.1 x lo6 cases each year. Despite antibiotic therapy the case fatality rate is about 5-7%, making pneumonia one of the major causes of morbidity and mortality in the developed world. In the same study, Austrian estimated that the attack rate for septicaemia was between 25 and 50 per 100000 population per annum, with a case fatality rate of between 17 and 24%. Most, but not all, of these cases were associated with lower respiratory tract infections. In an attempt to focus attention on the desirability of developing an effective vaccine to protect against pneumococcal infection, this situation was likened, in terms of incidence and mortality, to that of poliomyelitis prior to the introduction of the vaccine. The incidence of pneumococcal meningitis is estimated to be about 1.5 per 100000 population per annum (Austrian, 1982b). It seems likely that similar attack and fatality rates are

Journal ArticleDOI
TL;DR: According to their insertion sites the Tn5 mutants were mapped into several classes: auxotrophs, exopolysaccharide (EPS)-deficient mutants and a mutant of the dsp phenotype, which could complement each other to produce virulence symptoms on pear slices.
Abstract: SUMMARY: Transposon Tn5, on a mobilizable ColE1 plasmid, on a Ti plasmid derepressed for bacterial transfer, and on the bacteriophage fd genome, was used to construct pathogenicity mutants of the fire blight pathogen Erwinia amylovora. Eleven nonpathogenic mutants were isolated from 1600 independent mutants screened. These mutants were divided into three types: auxotrophs, exopolysaccharide (EPS)-deficient mutants and a mutant of the dsp phenotype. According to their insertion sites the Tn5 mutants were mapped into several classes. Some of the mutants could be complemented with cosmid clones from a genomic library of the parent strain for EPS production on minimal agar. EPS-deficient mutants and the dsp mutant could complement each other to produce virulence symptoms on pear slices.

Journal ArticleDOI
TL;DR: Partial 16S ribosomal ribonucleic acid sequences of five Aerococcus-like organisms originally isolated from patients with urinary tract infections were determined using reverse transcriptase in order to clarify their taxonomic position.
Abstract: SUMMARY: Partial 16S ribosomal ribonucleic acid sequences of five Aerococcus-like organisms originally isolated from patients with urinary tract infections were determined using reverse transcriptase in order to clarify their taxonomic position. Analysis of the sequence data revealed that the clinical isolates represent a hitherto unknown line of descent within the genus Aerococcus. A new species, Aerococcus urinae, is proposed for these isolates. The type strain is NCFB 2893.

Journal ArticleDOI
TL;DR: The dehalogenases H-1 and H-2 have no enzyme-evolutionary relationship, and the deduced amino acid sequence of the dehH1 gene showed significant similarity to those of three hydrolases of Pseudomonas putida and a haloalkane dehalagenase of Xanthobacter autotrophicus.
Abstract: Two genes encoding haloacetate dehalogenases, H-1 and H-2, are closely linked on a plasmid from Moraxella sp. strain B. H-1 predominantly acts on fluoroacetate, but H-2 does not. To elucidate the molecular relationship between the two enzymes, we compared their structural genes. Two restriction fragments of the plasmid DNA were subcloned on M13 phages and their nucleotide sequences were determined. The sequence of each fragment contained an open reading frame that was identified as the structural gene for each of the two dehalogenases on the basis of the following criteria; N-terminal amino acid sequence, amino acid composition, and molecular mass. The genes for H-1 and H-2, designated dehH1 and dehH2, respectively, had different sizes (885 bp and 675 bp) and G + C contents (58.3% and 53.4%). Sequence analysis revealed no homology between the two genes. We concluded that the dehalogenases H-1 and H-2 have no enzyme-evolutionary relationship. The deduced amino acid sequence of the dehH1 gene showed significant similarity to those of three hydrolases of Pseudomonas putida and a haloalkane dehalogenase of Xanthobacter autotrophicus. The dehH2 coding region was sandwiched between two repeated sequences about 1.8 kb long, which might play a part in the frequent spontaneous deletion of dehH2 from the plasmid.

Journal ArticleDOI
TL;DR: The storage of iron within bacteria is also important, particularly in properly tuning the flux of iron required by iron-proteins involved, and has been actively studied in the last decade.
Abstract: Iron is an essential element for most living organisms. Its roles in microbial physiology are numerous. Iron is a constituent of all haem enzymes, which include cytochromes and hydroperoxidases. The common type of ribonucleotide reductase contains iron, and non-iron nitrogenases require an iron protein in a complex for their activity (Robson et al., 1986). However, exceptions do exist; for example, certain lactobacilli devoid of haem and containing a cobalt form of ribonucleotide reductase appear to have no iron requirement for their growth (Archibald, 1983). Iron, the fourth most-represented element in the earth’s crust, is abundant in the environment and should not be a limiting factor for bacterial growth. However, in the presence of oxygen and at a non-acidic pH, iron is particularly insoluble and tends to precipitate as ferric hydroxides. Therefore, bacteria have evolved various powerful systems to overcome this low solubility of external iron (Lankford, 1973). Besides its insolubility, another problem associated with the metabolism of iron resides in its ability to react with reduced forms of oxygen (hydrogen peroxide and superoxide), leading to the production of deleterious free radicals responsible for lipid peroxidation, as well as for alterations in protein and nucleic acids (Flitter et al., 1983). Therefore, in order to avoid such toxicity, iron homeostasis is strictly controlled and results from a co-ordinated integration of assimilation, utilization and storage of this element. Iron uptake is an obvious step to be regulated in response to variations in environmental iron concentration, and has been actively studied in the last decade. The first part of this paper is a non-exhaustive overview of iron assimilation and its control in prokaryotes. The storage of iron within bacteria is also important, particularly in properly tuning the flux of iron required by iron-proteins involved

Journal ArticleDOI
TL;DR: Six biodegradative actinomycete strains were grown on a dimeric model lignin compound andExtracellular peroxidase and catalase activity were detected in all of the strains and are used to propose a scheme by which actinologycete attack of the lign in component of plant biomass can be envisaged.
Abstract: Six biodegradative actinomycete strains were grown on a dimeric model lignin compound of the β-aryl ether type. Although only two strains, Thermomonospora mesophila and Streptomyces badius, utilized the compound as a carbon and energy source and produced substantial amounts of monomeric products, all of the strains could demethylate the substrate and oxidize Cα on the phenylpropane side-chain. Streptomyces sp. EC1 produced small amounts of aromatic acids and unidentified lignin-derived products when grown on straw. This organism also produced cell-bound demethylase requiring H2O2 and Mn2+, protocatechuate 3,4-dioxygenase and β-carboxymuconate decarboxylase activity in response to growth on low-molecular-mass aromatic compounds but not lignocellulose or its polysaccharide components. Extracellular peroxidase and catalase activity were detected in all of the strains. These data are used to propose a scheme by which actinomycete attack of the lignin component of plant biomass can be envisaged.

Journal ArticleDOI
TL;DR: Highly larvicidal strains of Bacillus sphaericus produce a binary toxin which binds to sharply delineated regions of the gastric caecum and posterior midgut of susceptible larvae of the mosquito Culex quinquefasciatus and toxicity was strongly correlated with the subsequent internalization of the toxin, probably by endocytosis.
Abstract: Highly larvicidal strains of Bacillus sphaericus produce a binary toxin composed of 51 and 42 kDa proteins which binds to sharply delineated regions of the gastric caecum and posterior midgut of susceptible larvae of the mosquito Culex quinquefasciatus. To investigate the role of the individual subunits and the organization of functional binding regions within the toxin, plasmids were constructed for the expression in Escherichia coli of the toxin proteins and their NH2- and COOH-terminal deletion derivatives as fusions with glutathione S-transferase (GST). Toxin proteins were purified by affinity chromatography followed by cleavage from the GST carrier with thrombin. The LC50 values for the purified toxin proteins and their deletion derivatives were determined. The binding patterns of fluorescently labelled toxin suggested that the 51 kDa protein is the primary binding component of the toxin and mediates the regional binding and internalization of the 42 kDa protein. Examination of the toxin deletion derivatives revealed that the NH2-terminal region of the 51 kDa protein was required for binding to the larval gut, whilst the COOH-terminal region was responsible for interacting with the 42 kDa protein. Toxicity was strongly correlated with the subsequent internalization of the toxin, probably by endocytosis.

Journal ArticleDOI
TL;DR: Fingerprint patterns were generated from strains of Neisseria meningitidis by digestion of chromosomal DNA samples with 'rare-site' restriction endonucleases and resolution of the resultant fragments by pulsed field gel electrophoresis (PFGE).
Abstract: Summary: Fingerprint patterns were generated from strains of Neisseria meningitidis by digestion of chromosomal DNA samples with ‘rare-site’ restriction endonucleases and resolution of the resultant fragments by pulsed field gel electrophoresis (PFGE). The potential of this technique for the rapid establishment of the clonal relationships between different isolates of the meningococcus was investigated. The fingerprint patterns from various serogroup A strains, previously assigned to clonal subgroups on the basis of their electrophoretic types (ETs), were compared. Fingerprints generated with the endonucleases SfiI, SpeI and NheI each gave distinctive patterns for the clonal subgroups I-IV of serogroup A. Further, the endonucleases Spel and, particularly, NheI were capable of resolving differences between various subgroup III strains isolated at different times and geographical locations. Strains isolated during the ‘new wave’ pandemic, which was associated with the Haj, from Europe, America, and Africa, had a characteristic fingerprint pattern and appeared to be distinct from ‘old wave’ pandemic strains. The PFGE technique is a relatively rapid and sensitive method for establishing clonal relationships among epidemic strains of N. meningitidis.

Journal ArticleDOI
TL;DR: It is likely that the control of uhpT transcription by catabolite repression exists to limit the level of UhpT transport activity and thereby prevent the toxic events that result from elevated uptake of its substrates.
Abstract: The Escherichia coli uhpT gene encodes an active transport system for sugar phosphates. When the uhpT gene was carried on a multicopy plasmid, amplified levels of transport activity occurred, and growth of these strains was inhibited upon the addition of various sugar phosphates. Two different mechanisms for this growth inhibition were distinguished. Exposure to glucose-6-phosphate, fructose-6-phosphate or mannose-6-phosphate, which enter directly into the glycolytic pathway, resulted in cessation of growth and substantial loss of viability. Cell killing was correlated with the production of the toxic metabolite, methylglyoxal. In contrast, addition of 2-deoxyglucose-6-phosphate, galactose-6-phosphate, glucosamine-6-phosphate or arabinose-5-phosphate, which do not directly enter the glycolytic pathway, resulted in growth inhibition without engendering methylglyoxal production or cell death. Inhibition of growth could result from excessive accumulation of organophosphates in the cell or depletion of inorganic phosphate pools as a result of the sugar-P/Pi exchange process catalysed by UhpT. The phosphate-dependent uptake of glycerol-3-phosphate by the GlpT antiporter was strongly inhibited under conditions of elevated sugar-phosphate transport. There are thus two separate toxic effects of elevated sugar-phosphate transport, one of which was lethal and related to increased flux through glycolysis. It is likely that the control of uhpT transcription by catabolite repression exists to limit the level of UhpT transport activity and thereby prevent the toxic events that result from elevated uptake of its substrates.

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TL;DR: It is found that, in the blood of infected animals, the protective antigen exists primarily as a 63 kDa protein and appears to be complexed with the lethal factor component of the toxin.
Abstract: Summary: The protective antigen component of anthrax lethal toxin, produced in vitro, has a molecular mass of 83 kDa. Cell-culture studies by others have demonstrated that upon binding of the 83 kDa protective antigen to cell-surface receptors, the protein is cleaved by an unidentified cell-associated protease activity. The resultant 63 kDa protein then binds lethal factor to form lethal toxin, which has been proposed to be internalized by endocytosis. We found that, in the blood of infected animals, the protective antigen exists primarily as a 63 kDa protein and appears to be complexed with the lethal factor component of the toxin. Conversion of protective antigen from 83 to 63 kDa was catalysed by a calcium-dependent, heat-labile serum protease. Except for being complexed to protective antigen, there was no apparent alteration of lethal factor during the course of anthrax infection. The protective antigen-cleaving protease appeared to be ubiquitous among a wide range of animal species, including primates, horses, goats, sheep, dogs, cats and rodents.

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TL;DR: Homologous schemes of secondary structure were deduced for precursor rRNA of both M. tuberculosis and M. leprae; although the principal features are common to both species there are notable differences.
Abstract: Mycobacterium tuberculosis H37Rv has a single rrn (ribosomal RNA) operon. The operon was cloned and a region of 1536 nucleotides was sequenced, starting 621 bp upstream from the 5'-end of the 16S rRNA coding region and continuing to the start of the 23S rRNA coding region. The 16S rRNA sequence inferred from the gene sequence was found to differ in one position from Mycobacterium bovis (nucleotide 1443) and from Mycobacterium microti (nucleotide 427). A single putative promoter was identified on the basis of similarities with the sequence of rrn operons of Bacillus subtilis and Escherichia coli. The regions of similarity include a — 35 box, a — 10 box, a stringent response element, antitermination signals, potential RNAase III processing sites and features of prefnsor rRNA secondary structure. Sequences upstream from the 5'-end of Mycobacterium leprae 16S rRNA were also investigated. Homologous schemes of secondary structure were deduced for prefnsor rRNA of both M. tuberculosis and M. leprae; although the principal features are common to both species there are notable differences.

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TL;DR: Results are discussed in terms of the metabolic significance of PHB synthesis in cyanobacteria, and it is suggested that this polymer acts exclusively as a disposal mechanism to eliminate excess reducing equivalents.
Abstract: The effect of different growth conditions on the glycogen and poly-β-hydroxybutyrate (PHB) content of the cyanobacterium Spirulina maxima is described. Under photoautotrophic growth conditions without any nutrient limitation, S. maxima exhibited a glycogen content of between 7.1 and 10.7% of cell dry wt, whereas PHB was undetectable. When S. maxima was grown under mixotrophic conditions in the presence of acetate, the intracellular PHB concentration increased to more than 3% of dry wt, while glycogen content remained within the range of 5 to 6% of cell dry wt. Nitrogen starvation favoured glycogen accumulation (up to 60 to 70% of dry wt), while the PHB content remained low (up to 0.7% of dry wt), even after prolonged nitrogen starvation. Inhibition of protein synthesis, induced by addition of azaserine, led to the accumulation of glycogen (up to 52% of cell dry wt) but did not stimulate PHB synthesis. Under phosphorus-limited growth conditions, glycogen and PHB accumulated (up to 23% and 1.2% of cell dry wt, respectively) only after the exhaustion of intracellular phosphorus reserves. Shifting the culture from low to high light irradiance induced a rapid accumulation of glycogen (up to 34% of cell dry wt after 9 h) but did not induce PHB synthesis. Results are discussed in terms of the metabolic significance of PHB synthesis in cyanobacteria, and suggest that this polymer acts exclusively as a disposal mechanism to eliminate excess reducing equivalents.