Antibiotic activity and siderophores of Pseudomonas cepacia

The ability of 46 strains of Pseudomonas cepacia to inhibit phytopathogenic fungi and the effect of iron on their antifungal activity were studied. The antifungal effect of the bacteria and the antimicrobial activity of their crude yellow and violet pigments showed a 4-5-fold decrease in the presence of Fe(III). The addition of 100 micrograms/ml of FeCl3 to the medium decreased the biosynthesis of violet and yellow pigments; the complex of the yellow pigment with Fe(III) promoted the growth of the P. cepacia producing strain under iron-deficient conditions. The data obtained suggest a participation of some P. cepacia pigments in iron transport. The resistance of the P. cepacia strains to the synthetic chelating agents hydroxyethylenediphosphonic and diethylenediaminepentaacetic acids was demonstrated, which may indicate a high Fe(III)-binding constant of P. cepacia siderophores.     

Bacteriocin typing of Pseudomonas cepacia strains isolated from patients and the rhizosphere of plants

The work deals with the bacteriocin typing of 34 P. cepacia strains isolated from different sources with respect to both the capacity of synthesizing bactericins and sensitivity to them. The standard set of strains comprizing 8 P. cepacia bacteriocin-sensitive strains and 6 highly active cepaciacin producer strains was used. 24 P. cepacia strains belonged to 11 different S-types, 20 strains synthetized cepaciacins of new types.     

Production kinetics of acidophilin 801, a bacteriocin produced by Lactobacillus acidophilus IBB 801

Lactobacillus acidophilus IBB 801 produces a small bacteriocin, designated acidophilin 801. Studying the relationship between growth and bacteriocin biosynthesis revealed primary metabolite kinetics of bacteriocin production with a peak activity at the end of the exponential growth phase followed by a decrease during the stationary phase. Both microbial growth and bacteriocin production was inhibited by lactic acid. Whereas volumetric bacteriocin production (activity units (AU) ml(-1)) was favoured under pH-controlled conditions, bacteriocin titres rapidly decreased because of strong adsorption of the bacteriocin molecules to the producing cells under less acidic conditions.     

Bacteriocin, plasmid and pectolytic diversity in Pseudomonas cepacia of clinical and plant origin.

Pseudomonas cepacia strains of plant and clinical origin were compared with the type strains of P. cepacia, P. kingii and P. multivorans. Conventional biochemical tests and antibiotic sensitivity patterns supported the previous proposals of synonymy between P. cepacia, P. kingii and P. multivorans. However, bacteriocin production patterns, onion maceration tests and hydrolysis of low pH pectate agar clearly differentiated strains of clinical and plant origin into two distinct groups; these tests may therefore be helpful in epidemiological studies. In contrast, plant and clinical strains were of equal lethality to mice. Agarose gel electrophoresis indicated the presence of one or more plasmids (molecular weights 9 X 10(6) to 120 X 10(6)) in 15 out of 16 strains of both types examined.     

Sensitivity of the Burkholderia cepacia complex and Pseudomonas aeruginosa to transducing bacteriophages

Burkholderia cepacia is now recognised as a life-threatening pathogen among several groups of immunocompromised patients. In this context, the proposed large-scale use of these bacteria in agriculture has increased the need for a better understanding of the genetics of the species forming the B. cepacia complex. Until now, little information has been available on the bacteriophages of the B. cepacia complex. Transducing phages, named NS1 and NS2, were derived from the lysogenic B. cepacia strains ATCC 29424 and ATCC 17616. The frequency of transduction per phage particle ranged from 1.0x10(-8) to 7.0x10(-6) depending on the phage and recipient strain used. The host range of NS1 and NS2 differed but in each case included environmental and clinical isolates, and strains belonging to several species and genomovars of the B. cepacia complex. The host range of both phages also included Pseudomonas aeruginosa. Some B. cepacia complex isolates were sensitive to the well-characterised P. aeruginosa transducing phages, B3, F116L and G101. The lytic activity of NS1 and NS2 was inhibited by B. cepacia lipopolysaccharide suggesting that this moiety is a binding site for both phages. The molecular size of the NS1 and NS2 genomes was approximately 48 kb.     

Characterization of a proteinaceous antimicrobial produced by Lactobacillus helveticus CNRZ450

An antimicrobial substance which resembles a bacteriocin was identified in culture supernatant fluids of Lactobacillus helveticus strain CNRZ450. The bacteriocin was active against a narrow range of strains from closely rested species of homofermentative lactobacilli. Its mode of action appeared to be bacteriostatic. Partial purification of the bacteriocin suggested that it was a complex protein with a mol. wt of between 30 and 50 kDa, although there is some evidence that the polypeptide monomer has a mol. wt of around 17 kDa. There was no evidence indicating an extrachromosomal location for its genetic determinant. PCR generated an amplicon from total DNA from strain CNRZ450 using primers based on the helJ gene sequence. A fragment showing homology to this amplicon was located in an Eco RI digest of total DNA from strain CNRZ450. The pattern obtained was different from that obtained with the helveticin J producer strain NCFB481. It is possible, therefore, that the antimicrobial from strain CNRZ450 is related to helveticin J at the DNA sequence level although the physical properties of the two antimicrobials reveal several differences.     

Purification and characterization of a complex between cloacin and its immunity protein isolated from Enterobacter cloacae (Clo DF13). Dissociation and reconstitution of the complex.

Cell of Enterobacter cloacae (Clo DF13) produce a bacteriocin which is characterized by its very effective killing activity against sensitive bacteria. Purification and characterization of the excreted bacteriocin has revealed that this bacteriocin consists of an equimolar complex of two plasmid-specific gene products: the cloacin and its inhibitor the immunity protein. Dissociation of the complex by treatment with sodium dodecylsulfate induces the endonucleolytic activity of the cloacin but strongly reduces the killing activity. The purified complex possesses no activity in vitro. Both cloacin and immunity protein isolated from the complex were functionally identical to cloacin and immunity protein purified from the bacteriocinogenic cells by other methods. Reconstitution of the complex results in a partial restoration of killing activity.     

Complex of Burkholderia cepacia lipase with transition state analogue of 1-phenoxy-2-acetoxybutane: biocatalytic, structural and modelling study.

In a series of four racemic phenoxyalkyl-alkyl carbinols, 1-phenoxy-2-hydroxybutane (1) is enantioselectively acetylated by Burkholderia cepacia (formerly Pseudomonas cepacia) lipase with an E value > or = 200, whereas for the other three racemates E was found to be < or = 4. To explain the high preference of B. cepacia lipase for (R)-(+)-1, a precursor of its transition state analogue with a tetrahedral P-atom, (R(P),S(P))-O-(2R)-(1-phenoxybut-2-yl)methylphosphonic acid chloride was prepared and crystallized in complex with B. cepacia lipase. The X-ray structure of the complex was determined, allowing to compare the conformation of the inhibitor with results of molecular modelling.     

Bacteriocin-Producing Strain of Lactococcus lactis subsp. diacitilactis S50

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

Functional Genetic Analysis of the GarML Gene Cluster in Lactococcus garvieae DCC43 Gives New Insights into Circular Bacteriocin Biosynthesis

Garvicin ML (GarML) is a circular bacteriocin produced by Lactococcus garvieae DCC43. The recently published draft genome of this strain allowed determination of the genetic background for bacteriocin production. Bioinformatic analysis identified a gene cluster consisting of nine open reading frames likely involved in the production of and immunity to GarML. The garA gene encodes the bacteriocin precursor, garX a large transmembrane protein, garBCDE a putative immunity protein (garB) followed by an ATPase and two transmembrane proteins, and garFGH a putative ABC transporter complex. Functional genetic analysis revealed that deletion of garFGH had no effect on sensitivity to or production of GarML. In contrast, deletion of garBCDE or inactivation of garX resulted in high-level sensitivity to GarML and completely abolished production of active bacteriocin. Mass spectrometry of culture supernatants revealed that wild-type cultures contained the mature circular form as well as the linear forms of the bacteriocin, both with and without the three-amino-acid leader sequence, while bacteriocin-negative mutants contained only the linear forms. These results indicate that cleavage of the leader peptide precedes circularization and is likely performed by a functional entity separate from the GarML gene cluster. To our knowledge, this is the first conclusive evidence for these processes being separated in time. Loss of immunity and antimicrobial activity in addition to our inability to detect the circular bacteriocin in the ΔgarBCDE and garX::pCG47 mutants demonstrate that both these units are indispensable for GarML biosynthesis as well as immunity. Furthermore, the results indicate that these genes are implicated in the circularization of the bacteriocin and that their functions are probably interlinked.     

Interspecies biofilms of Pseudomonas aeruginosa and Burkholderia cepacia.

The leading cause of morbidity and mortality in cystic fibrosis (CF) continues to be lung infections with Pseudomonas aeruginosa biofilms. Co-colonization of the lungs with P aeruginosa and Burkholderia cepacia can result in more severe pulmonary disease than P. aeruginosa alone. The interactions between P. aeruginosa biofilms and B. cepacia are not yet understood; one possible association being that mixed species biofilm formation may be part of the interspecies relationship. Using the Calgary Biofilm Device (CBD), members of all genomovars of the B. cepacia complex were shown to form biofilms, including those isolated from CF lungs. Mixed species biofilm formation between CF isolates of P. aeruginosa and B. cepacia was readily achieved using the CBD. Oxidation-fermentation lactose agar was adapted as a differential agar to monitor mixed biofilm composition. Scanning electron micrographs of the biofilms demonstrated that both species readily integrated in close association in the biofilm structure. Pseudomonas aeruginosa laboratory strain PAO1, however, inhibited mixed biofilm formation of both CF isolates and environmental strains of the B. cepacia complex. Characterization of the soluble inhibitor suggested pyocyanin as the active compound.     

Stereoselectivity of Pseudomonas cepacia lipase toward secondary alcohols: a quantitative model

The lipase from Pseudomonas cepacia represents a widely applied catalyst for highly enantioselective resolution of chiral secondary alcohols. While its stereopreference is determined predominantly by the substrate structure, stereoselectivity depends on atomic details of interactions between substrate and lipase. Thirty secondary alcohols with published E values using P. cepacia lipase in hydrolysis or esterification reactions were selected, and models of their octanoic acid esters were docked to the open conformation of P. cepacia lipase. The two enantiomers of 27 substrates bound preferentially in either of two binding modes: the fast-reacting enantiomer in a productive mode and the slow-reacting enantiomer in a nonproductive mode. Nonproductive mode of fast-reacting enantiomers was prohibited by repulsive interactions. For the slow-reacting enantiomers in the productive binding mode, the substrate pushes the active site histidine away from its proper orientation, and the distance d(H(N epsilon) - O(alc)) between the histidine side chain and the alcohol oxygen increases, d(H(N epsilon) - O(alc)) was correlated to experimentally observed enantioselectivity: in substrates for which P. cepacia lipase has high enantioselectivity (E > 100), d(H(N epsilon) - O(alc)) is >2.2 A for slow-reacting enantiomers, thus preventing efficient catalysis of this enantiomer. In substrates of low enantioselectivity (E < 20), the distance d(H(N epsilon) - O(alc)) is less than 2.0 A, and slow- and fast-reacting enantiomers are catalyzed at similar rates. For substrates of medium enantioselectivity (20 < E < 100), d(H(N epsilon) - O(alc)) is around 2.1 A. This simple model can be applied to predict enantioselectivity of P. cepacia lipase toward a broad range of secondary alcohols.     

Purification and characterization of a bacteriocin produced by Lactobacillus acidophilus IBB 801

Lactobacillus acidophilus IBB 801 produces a small bacteriocin, designated acidophilin 801, with an estimated molecular mass of less than 6.5 kDa. It displays a narrow inhibitory spectrum (only related lactobacilli but including the Gram-negative pathogenic bacteria Escherichia coli Row and Salmonella panama 1467) with a bactericidal activity. The antimicrobial activity of cell-free culture supernatant fluid was insensitive to catalase but sensitive to proteolytic enzymes such as trypsin, proteinase K and pronase, heat-stable (30 min at 121 degrees C), and maintained in a wide pH range. The proteinaceous compound was isolated from cell-free culture supernatant fluid and purified. Crude bacteriocin was isolated as a floating pellicle after ammonium sulphate precipitation (40% saturation) and partially purified by extraction/precipitation with chloroform/methanol (2/1, v/v). Further purification to homogeneity was performed by reversed phase Fast Performance Liquid Chromatography. The amino acid composition was determined. Amino acid sequencing revealed that the N-terminal end was blocked.     

The murI gene of Escherichia coli is an essential gene that encodes a glutamate racemase activity.

The murI gene of Escherichia coli was recently identified on the basis of its ability to complement the only mutant requiring D-glutamic acid for growth that had been described to date: strain WM335 of E. coli B/r (P. Doublet, J. van Heijenoort, and D. Mengin-Lecreulx, J. Bacteriol. 174:5772-5779, 1992). We report experiments of insertional mutagenesis of the murI gene which demonstrate that this gene is essential for the biosynthesis of D-glutamic acid, one of the specific components of cell wall peptidoglycan. A special strategy was used for the construction of strains with a disrupted copy of murI, because of a limited capability of E. coli strains grown in rich medium to internalize D-glutamic acid. The murI gene product was overproduced and identified as a glutamate racemase activity. UDP-N-acetylmuramoyl-L-alanine (UDP-MurNAc-L-Ala), which is the nucleotide substrate of the D-glutamic-acid-adding enzyme (the murD gene product) catalyzing the subsequent step in the pathway for peptidoglycan synthesis, appears to be an effector of the racemase activity.     

Characterization of a partially purified bacteriocin, Fermentcin B, from Lactobacillus fermentum

A bacteriocin, Fermentcin B produced by Lactobacillus fermentum, was identified from the inhibitory products of twenty-nine mesophilic lactic acid bacteria. It has a bactericidal activity with a narrow inhibitory spectrum. The bacte-riocin is heat stable at 100° 30 min and stable in pH range of 3.0 to 8.0. Fermentcin B lost its activity after treatment with a-chymotrypsin, proteinase-K, and amyloglucosidase.     

Isolation and partial characterization of a bacteriocin produced by Lactobacillus plantarum BM‐1 isolated from a traditionally fermented Chinese meat product

Lactobacillus plantarum BM‐1 isolated from a traditionally fermented Chinese meat product was found to produce a novel bacteriocin that is active against a wide range of gram‐positive and gram‐negative bacteria. Production of the bacteriocin BM‐1 started early in the exponential phase and its maximum activity (5120 AU/mL) was recorded early during the stationary phase (16 hr). Bacteriocin BM‐1 is sensitive to proteolytic enzymes but stable in the pH range of 2.0–10.0 and heat‐resistant (15 min at 121°C). This bacteriocin was purified through pH‐mediated cell adsorption–desorption and cation‐exchange chromatography on an SP Sepharose Fast Flow column. The molecular weight of the purified bacteriocin BM‐1 was determined to be 4638.142 Da by electrospray ionization Fourier transform mass spectrometry. Furthermore, the N‐terminal amino acid sequence was obtained through automated Edman degradation and found to comprise the following 15 amino acid residues: H₂N‐Lys‐Tyr‐Tyr‐Gly‐Asn‐Gly‐Val‐Tyr‐Val‐Gly‐Lys‐His‐Ser‐Cys‐Ser. Comparison of this sequence with that of other bacteriocins revealed that bacteriocin BM‐1 contains the consensus YGNGV amino acid motif near the N‐terminus. Based on its physicochemical characteristics, molecular weight, and N‐terminal amino acid sequence, plantaricin BM‐1 is a novel class IIa bacteriocin.     

Identification of N-acylhomoserine lactones in mucopurulent respiratory secretions from cystic fibrosis patients

Pseudomonas aeruginosa and species of the Burkholderia cepacia complex are the primary bacterial pathogens contributing to lung disease in patients with cystic fibrosis. Quorum sensing systems using N-acyl homoserine lactone (AHL) signal molecules are involved in the regulation of a number of virulence factors in these species. Extracts of mucopurulent respiratory secretions from 13 cystic fibrosis patients infected with P. aeruginosa and/or strains of the B. cepacia complex were fractionated using reverse-phase fast pressure liquid chromatography and analyzed for the presence of AHLs using a traI-luxCDABE-based reporter that responds to AHLs with acyl chains ranging between 4 and 12 carbons. Using this assay system, a broad range of AHLs were detected and identified despite being present at low concentrations in limited sample volumes. N-(3-oxo-dodecanoyl)-l-homoserine lactone, N-(3-oxo-decanoyl)-l-homoserine lactone and N-octanoyl-l-homoserine lactone (OHL) were the AHLs most frequently identified. OHL and N-decanoyl-l-homoserine lactone were detected in nanomolar concentrations compared to picomolar amounts of the 3-oxo-derivatives of the AHLs identified.     

Characterization, N-terminal sequencing and classification of cerein MRX1, a novel bacteriocin purified from a newly isolated bacterium: Bacillus cereus MRX1

AIM: To purify and characterize the bacteriocin produced by strain MRX1. METHODS AND RESULTS: A bacteriocin-producing strain was isolated and identified as Bacillus cereus. The bacteriocin, called cerein MRX1, was purified from the culture supernatant using hydrophobic interaction, cation-exchange chromatography and RP-HPLC. It could also be purified in abundance from the cell surfaces of the producer strain. Mass spectrometry revealed its molecular mass of 3137.93 Da. Sequencing of chemically modified bacteriocin identified its partial sequence: DWTCWSCLVCAACSVELL. Amino acid analysis, confirmed by (1)H-NMR, suggested cerein MRX1 to be a class II bacteriocin. This bacteriocin was remarkably hydrophobic, heat-stable and could withstand a wide range of pH. It exhibited a bactericidal mode of action against Bacillus coagulans JCM 2257(T). Cerein MRX1 was especially active against spoilage bacteria such as Bacillus subtilis and Listeria innocua (MICs in the 1 microg ml(-1) range). In contrast, lactic acid bacteria were resistant or required higher concentrations to be inhibited. CONCLUSIONS: Cerein MRX1 is similar by its N-terminal sequence to thuricin 17 recently isolated from Bacillus thuringiensis NEB17. However, the two bacteriocins are different by their molecular masses and amino acid compositions. SIGNIFICANCE AND IMPACT OF THE STUDY: Chemical stability of cerein MRX1 and its ability to inhibit a large number of undesirable bacteria may give an advantage to its food or clinical application as an antibacterial agent.     

Identification, purification and characterization of laterosporulin, a novel bacteriocin produced by Brevibacillus sp. strain GI-9

Background

Bacteriocins are antimicrobial peptides that are produced by bacteria as a defense mechanism in complex environments. Identification and characterization of novel bacteriocins in novel strains of bacteria is one of the important fields in bacteriology.

Methodology/Findings

The strain GI-9 was identified as Brevibacillus sp. by 16 S rRNA gene sequence analysis. The bacteriocin produced by strain GI-9, namely, laterosporulin was purified from supernatant of the culture grown under optimal conditions using hydrophobic interaction chromatography and reverse-phase HPLC. The bacteriocin was active against a wide range of Gram-positive and Gram-negative bacteria. MALDI-TOF experiments determined the precise molecular mass of the peptide to be of 5.6 kDa and N-terminal sequencing of the thermo-stable peptide revealed low similarity with existing antimicrobial peptides. The putative open reading frame (ORF) encoding laterosporulin and its surrounding genomic region was fished out from the draft genome sequence of GI-9. Sequence analysis of the putative bacteriocin gene did not show significant similarity to any reported bacteriocin producing genes in database.

Conclusions

We have identified a bacteriocin producing strain GI-9, belonging to the genus Brevibacillus sp. Biochemical and genomic characterization of laterosporulin suggests it as a novel bacteriocin with broad spectrum antibacterial activity.     

An Escherichia coli gene in search of a function: phenotypic effects of the gene recently identified as murI.

Earlier we reported that an open reading frame located at 89.5 min of the Escherichia coli map (ORFI) codes for a protein of unknown function that could be overexpressed and purified to homogeneity (G. Balikó, A. Raukas, I. Boros, and P. Venetianer, Mol. Gen. Genet. 211:326-331, 1988). In the work described here, we attempted to learn the function of this protein by inactivating the chromosomal gene and providing it or its deletion derivatives on temperature-sensitive plasmids. We found that the presence of the functional ORFI gene is essential; cells are not viable at the nonpermissive temperature or when the region coding for the C-terminal 50 amino acids of the protein is deleted. At intermediate temperatures or when the gene is overexpressed, characteristic changes occur in cell morphology, nucleoid separation during cell division, and supercoiling of plasmids. The possible mechanisms of these effects are discussed in view of the fact that Doublet et al. (P. Doublet, J. van Heijenoort, and D. Mengin-Lecreulx, J. Bacteriol. 174:5772-5779, 1992) recently identified the ORFI gene as murI, involved in D-glutamic acid biosynthesis.