Showing papers by "Todd R. Klaenhammer published in 1991"

Nisin treatment for inactivation of Salmonella species and other gram-negative bacteria.

Abstract: Nisin, produced by Lactococcus lactis subsp. lactis, has a broad spectrum of activity against gram-positive bacteria and is generally recognized as safe in the United States for use in selected pasteurized cheese spreads to control the outgrowth and toxin production of Clostridium botulinum. This study evaluated the inhibitory activity of nisin in combination with a chelating agent, disodium EDTA, against several Salmonella species and other selected gram-negative bacteria. After a 1-h exposure to 50 micrograms of nisin per ml and 20 mM disodium EDTA at 37 degrees C, a 3.2- to 6.9-log-cycle reduction in population was observed with the species tested. Treatment with disodium EDTA or nisin alone produced no significant inhibition (less than 1-log-cycle reduction) of the Salmonella and other gram-negative species tested. These results demonstrated that nisin is bactericidal to Salmonella species and that the observed inactivation can be demonstrated in other gram-negative bacteria. Applications involving the simultaneous treatment with nisin and chelating agents that alter the outer membrane may be of value in controlling food-borne salmonellae and other gram-negative bacteria.

Purification and partial characterization of lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088.

Abstract: Lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088 (NCK88), was purified and characterized. Lactacin F is heat stable, proteinaceous, and inhibitory to other lactobacilli as well as Enterococcus faecalis. The bacteriocin was isolated as a floating pellet from culture supernatants brought to 35 to 40% saturation with ammonium sulfate. Native lactacin F was sized at approximately 180 kDa by gel filtration. Column fractions having lactacin F activity were examined by electron microscopy and contained micelle-like globular particles. Purification by ammonium sulfate precipitation, gel filtration, and high-performance liquid chromatography resulted in a 474-fold increase in specific activity of lactacin F. The purified bacteriocin was identified as a 2.5-kDa peptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lactacin F peptide retained activity after extraction from SDS-PAGE gel slices, confirming the identity of the 2.5-kDa peptide. Variants of NCK88 that failed to exhibit lactacin F activity did not produce the 2.5-kDa band. Sequence analysis of purified lactacin F identified 25 N-terminal amino acids containing an arginine residue at the N terminus. Composition analysis indicates that lactacin F may contain as many as 56 amino acid residues.

Cloning, phenotypic expression, and DNA sequence of the gene for lactacin F, an antimicrobial peptide produced by Lactobacillus spp.

Abstract: Lactacin F is a heat-stable bacteriocin produced by Lactobacillus acidophilus 11088. A 63-mer oligonucleotide probe deduced from the N-terminal lactacin F amino acid sequence was used to clone the putative laf structural gene from plasmid DNA of a lactacin F-producing transconjugant, L. acidophilus T143. One clone, NCK360, harbored a recombinant plasmid, pTRK160, which contained a 2.2-kb EcoRI fragment of the size expected from hybridization experiments. An Escherichia coli-L. acidophilus shuttle vector was constructed, and a subclone (pTRK162) containing the 2.2-kb EcoRI fragment was introduced by electroporation into two lactacin F-negative strains, L. acidophilus 89 and 88-C. Lactobacillus transformants containing pTRK162 expressed lactacin F activity and immunity. Bacteriocin produced by the transformants exhibited an inhibitory spectrum and heat stability identical to those of the wild-type bacteriocin. An 873-bp region of the 2.2-kb fragment was sequenced by using a 20-mer degenerate lactacin F-specific primer to initiate sequencing from within the lactacin F structural gene. Analysis of the resulting sequence identified an open reading frame which could encode a protein of 75 amino acids. The 25 N-terminal amino acids for lactacin F were identified within the open reading frame along with an N-terminal extension, possibly a signal sequence. The lactacin F N-terminal sequence, through the remainder of the open reading frame (57 amino acids; 6.3 kDa), correlated extremely well with composition analyses of purified lactacin F which also predicted a size of 51 to 56 amino acid residues. Molecular characterization of lactacin F identified a small hydrophobic peptide that may be representative of a common bacteriocin class in lactic acid bacteria.

In vivo genetic exchange of a functional domain from a type II A methylase between lactococcal plasmid pTR2030 and a virulent bacteriophage

Abstract: The conjugative plasmid pTR2030 confers bacteriophage resistance to lactococci by two independent mechanisms, an abortive infection mechanism (Hsp+) and a restriction and modification system (R+/M+). pTR2030 transconjugants of lactococcal strains are used in the dairy industry to prolong the usefulness of mesophilic starter cultures. One bacteriophage which has emerged against a pTR2030 transconjugant is not susceptible to either of the two defense systems encoded by the plasmid. Phage nck202.50 (phi 50) is completely resistant to restriction by pTR2030. A region of homology between pTR2030 and phi 50 was subcloned, physically mapped, and sequenced. A region of 1,273 bp was identical in both plasmid and phage, suggesting that the fragment had recently been transferred between the two genomes. Sequence analysis confirmed that the transferred region encoded greater than 55% of the amino domain of the structural gene for a type II methylase designated LlaI. The LlaI gene is 1,869 bp in length and shows organizational similarities to the type II A methylase FokI. In addition to the amino domain, upstream sequences, possibly containing the expression signals, were present on the phage genome. The phage phi 50 fragment containing the methylase amino domain, designated LlaPI, when cloned onto the shuttle vector pSA3 was capable of modifying another phage genome in trans. This is the first report of the genetic exchange between a bacterium and a phage which confers a selective advantage on the phage. Definition of the LlaI system on pTR2030 provides the first evidence that type II systems contribute to restriction and modification phenotypes during host-dependent replication of phages in lactococci.

Rapid Method To Characterize Lactococcal Bacteriophage Genomes

Abstract: We present a rapid method to isolate and analyze bacteriophage DNA. Cells are infected and phage replication is allowed to proceed normally for 30 to 60 min. Prior to DNA packaging and cell bursts, the infected cells (1 ml) are harvested and lysed by using a combination of lysozyme and sodium dodecyl sulfate treatments. The total DNA recovered is enriched for phage genomes, and restriction fragments of the phage DNA can be readily visualized on agarose gels. This method was used to grossly compare the genomes of nine lactococcal phages isolated from different cheese plants at different times. The method was also used to visualize the inhibitory effects of pTR2030-induced abortive infection on the replication of phage nck202.31 in its homologous host, Lactococcus lactis NCK203.

Molecular Characterization of Three Small Isometric-Headed Bacteriophages Which Vary in Their Sensitivity to the Lactococcal Phage Resistance Plasmid pTR2030.

Abstract: Lactococcus lactis LMA12-4 is a pTR2030 transconjugant that has been used as an industrial starter culture because of its resistance to phages predominant in cheese plants Plasmid pTR2030 interferes with susceptible phages in this host strain via two mechanisms, restriction and modification (R/M) and abortive infection (Hsp) After prolonged use of LMA12-4 transconjugants in the industry, two different bacteriophages, designated nck202phi48 (phi48) and nck202phi50 (phi50), were isolated which could produce plaques on LMA12-4 containing pTR2030 In this study, these two phages were characterized and compared with a third phage, nck202phi31 (phi31), which is susceptible to both the R/M and Hsp activities encoded by pTR2030 Phage phi48 was not susceptible to inhibition by Hsp, whereas phi50 was unaffected by either the R/M or Hsp mechanisms All three were small isometric-headed phages, but small differences were noted between the phages in the structural details of the tail base plate, susceptibility to chloroform treatment, and requirements for calcium infectivity The phage genomes were all between 299 and 319 kb in length Phages phi31 and phi48 harbored cohesive ends, whereas the phage phi50 genome was circularly permuted, terminally redundant, and carried a putative packaging initiation site DNA-DNA hybridization experiments conducted between the phages revealed a common region in phi48 and phi50 that may correlate with the resistance of the two phages to the Hsp-abortive infection induced by pTR2030 Phage phi50 also harbored DNA sequences that shared homology to pTR2030 in the region where R/M activities have been localized on the plasmid Molecular characterization of the three phages localized regions within the genomes of the pTR2030-resistant phages that may be responsible for circumventing plasmid-encoded Hsp and R/M defense mechanisms in lactococci

Development of bacteriophage-resistant strains of lactic acid bacteria.

Abstract: One exciting area that has emerged through genetic analysis of the lactococci is the definition and practical application of gene systems that provide phage resistance to these industrially important bacteria. Naturally occurring phage-insensitive strains have been characterized and found to harbour multiple defence systems which can act at different points of the lytic cycle to prevent the successful adsorption, infection, or replication of virulent phages [ 11. Although phage attack on lactococcal starter cultures is still a major problem for the cultured dairy products industries, this can now be successfully addressed using genetic approaches to construct strains which are resistant to the phages often encountered in the industry [2, 31. In this paper I will describe the various phage defence systems that are naturally present in lactococci, their individual and combined effects, and the genetic strategies which are currently available to construct phageinsensitive strains for dairy fermentations. In addition, I will discuss the molecular responses of virulent phages which have appeared in the industry following the introduction and use of specialized starter cultures carrying defined mechanisms of phage resistance. The genetic routes whereby industrial phages elicit counter-defences against lactococcal resistance mechanisms provide new and important information that will facilitate the development of improved starter culture systems and phage-resistant lactic acid bacteria. Historically, starter strains that perform well in dairy fermentations have been selected on the basis of long-lived and consistent fermentative activity in phage-contaminated environments. Genetic analysis of Lactococcus lactis subsp. lactis and subsp. eremot6 strains over the past ten years has revealed that the lactococci harbour a variety of natural phage-defence systems. These include plasmid-encoded defences that interfere with phage adsorption, numerous DNA restriction and modification systems of different specificity, and undefined mechanisms that abort the phage infection after injection of phage DNA into the cell (for reviews see [4-61). Numerous phage-resistance plasmids encoding the above classes of resistance

Construction of an IS946-based composite transposon in Lactococcus lactis subsp. lactis.

Abstract: An artificial composite transposon was constructed based on the lactococcal insertion sequence IS946. A 3.0-kb element composed of the pC194 cat gene (Cmr) flanked by inversely repeated copies of IS946 was assembled on pBluescript KS+. When subcloned into the shuttle vector pSA3 (Emr), two putative transposons were created on the recombinant plasmid pTRK128: the 3.0-kb Cmr element (Tn-CmA) and an inverse 11.5-kb Emr element (Tn-EmA). pTRK128 was electroporated into the recombination-deficient strain Lactococcus lactis MMS362, which contains the self-transmissible plasmid pRS01. An MMS362 Cmr Emr transformant was used to assay for transposition events via conjugal mobilization of pTRK128-encoded Cmr or Emr to L. lactis LM2345. Transfer of either marker alone occurred at frequencies of ca. 2 x 10(-4) per input donor. Approximately 19% of the Emr transconjugants were Cms, indicating loss of the cat gene marker. No Cmr Ems transconjugants were recovered (n = 550). Plasmid analysis showed that the Cms Emr isolates contained a single large plasmid that was determined to be a cointegrate between pRS01 and the Tn-EmA element. A 32P-labeled pSA3 probe hybridized specifically to pTRK128 sequences and revealed different junction fragments within each of the cointegrate plasmids. DNA sequence analysis of the Tn-EmA::pRS01 junctions from a representative cointegrate verified transposition by Tn-EmA. This represents the first example of a functional composite transposon in the genus Lactococcus and serves as an experimental tool and model for the genetic analyses of transposons in these organisms. Images

Bacteriophage resistant recombinant bacteria

Abstract: Recombinant bacteria containing phage-encoded resistance ("Per") and methods of making and using the same are disclosed Such bacteria are made by (a) conducting a fermentation of a substrate in a medium containing a defined bacterial culture until bacteriophage are detected in the medium, the bacteriophage being specific to at least one bacteria in the medium; (b) isolating the bacteriophage; (c) digesting DNA of the bacteriophage to produce a library of DNA fragments; (d) transforming the bacteria susceptible to said bacteriophage with the library of DNA fragments to provide transformed bacteria; (e) selecting from among the transformed bacteria, a bacteriophage-resistant transformed bacteria; (f) adding bacteriophage resistant transformed bacteria to the medium; and (g) recommencing step (a) Also disclosed are bacterial cells which contain a first bacteriophage defense mechanism (Per), wherein Per comprises a bacteriophage origin of replication (ori) operatively associated with a DNA sequence incapable of producing live bacteriophage The bacterial cell is capable of being infected by a bacteriophage, the DNA of which, once injected into the bacterial cell, competes with Per for binding to DNA polymerase

Phage defense rotation strategy

Abstract: A phage defense rotation strategy for use in the successive fermentations of a substrate in a fermentation plant is disclosed. The strategy comprises (a) fermenting substrate with a first bacterial culture comprising a bacterial strain capable of fermenting the substrate and, preferably, carrying a first phage defense mechanism; and then (b) fermenting the substrate with a second bacterial culture comprising a second bacterial strain isogenic with the first bacterial strain, wherein the second strain carries a second phage defense mechanism different from the first phage defense mechanism. Also disclosed is a mixed bacterial culture capable of fermenting a substrate. The mixed culture comprises (a) a first bacterial strain carrying a first phage defense mechanism; and (b) a second bacterial strain isogenic with the first strain, wherein the second strain carries a phage defense mechanism different from the phage defense mechanism carried by the first strain.