Biomedical subjects

T R Klaenhammer

Publications and source records attributed to T R Klaenhammer.

At least 19 recordsLinked to original sources

PubMed2005

Sequence analysis of the Lactobacillus plantarum bacteriophage PhiJL-1.

The complete genomic sequence of a Lactobacillus plantarum virulent phage PhiJL-1 was determined. The phage possesses a linear, double-stranded, DNA genome consisting of 36,677 bp with a G+C content of 39.36%. A total of 52 possible open reading frames (ORFs) were identified. According to N-terminal amino acid sequencing and bioinformatic analyses, proven or putative functions were assigned to 21 ORFs (41%), including 5 structural protein genes. The PhiJL-1 genome shows functionally related genes clustered together in a genome structure composed of modules for DNA replication, DNA packaging, head and tail morphogenesis, and lysis. This type of modular genomic organization was similar to several other phages infecting lactic acid bacteria. The structural gene maps revealed that the order of the head and tail genes is highly conserved among the genomes of several Siphoviridae phages, allowing the assignment of probable functions to certain uncharacterized ORFs from phage PhiJL-1 and other Siphoviridae phages.

Amino Acid Sequence↗

PubMed2003

DNA analysis of the genes encoding acidocin LF221 A and acidocin LF221 B, two bacteriocins produced by Lactobacillus gasseri LF221.

Lactobacillus gasseri LF221, an isolate from the feces of a child, produces two bacteriocins. Standard procedures for molecular techniques were used to locate, clone and sequence the fragments of LF221 chromosomal DNA carrying the acidocin LF221 A and B structural genes, respectively. Sequencing analysis revealed the gene of acidocin LF221 A to be an open reading frame encoding a protein composed of 69 amino acids, including a 16-amino-acid N-terminal extension. The acidocin LF221 B gene was found to encode a 65-amino-acid bacteriocin precursor with a 17-amino-acid N-terminal leader peptide. DNA homology searches showed similarities of acidocin LF221 A to brochocin B, lactococcin N and thermophilin B, whereas acidocin LF221 B exhibited some homology to lactacin F and was virtually identical to gassericin X. The peptides encoded by orfA1 and orfB3 showed characteristics of class II bacteriocins and are suspected to be the complementary peptides of acidocin A and B, respectively. orfA3 and orfB5 are proposed to encode putative immunity proteins for the acidocins. Acidocin LF221 A and acidocin LF221 B are predicted to be members of the two-component class II bacteriocins, where acidocin LF221 A appears to be a novel bacteriocin. L. gasseri LF221 is being developed as a potential probiotic strain and a food/feed preservative. Detailed characterization of its acidocins is an important piece of background information useful in applying the strain into human or animal consumption. The genetic information on both acidocins also enables tracking of the LF221 strain in mixed populations and complex environments.

Amino Acid Sequence↗

PubMed2003

Isolation and characterization of a Lactobacillus plantarum bacteriophage, phiJL-1, from a cucumber fermentation.

A virulent Lactobacillus plantarum bacteriophage, PhiJL-1, was isolated from a commercial cucumber fermentation. The phage was specific for two related strains of L. plantarum, BI7 and its mutant (deficient in malolactate fermenting ability) MU45, which have been evaluated as starter cultures for controlled cucumber fermentation and as biocontrol microorganisms for minimally processed vegetable products. The phage genome of PhiJL-1 was sequenced to reveal a linear, double-stranded DNA (36.7 kbp). Sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE) profiles indicated that PhiJL-1 contains six structural proteins (28, 34, 45, 50, 61, and 76 kDa). Electron microscopy revealed that the phage has an isometric head (59 nm in diameter), a long non-contractile tail (182 nm in length and 11 nm in width), and a complex base plate. The phage belongs to the Bradley group B1 or Siphoviridae family. One-step growth kinetics of the phage showed that the latent period was 35 min, the rise period was 40 min, and the average burst size was 22 phage particles/infected cell. Phage particles (90%) adsorbed to the host cells 20 min after infection. Calcium supplementation (up to 30 mM CaCl(2)) in MRS media did not affect the first cycle of phage adsorption, but promoted rapid phage propagation and cell lysis in the infection cycle subsequent to adsorption. The D values of PhiJL-1 at pH 6.5 were estimated to be 2.7 min at 70 degrees C and 0.2 min at 80 degrees C by a thermal inactivation experiment. Knowledge of the properties of L. plantarum bacteriophage PhiJL-1 may be important for the development of controlled vegetable fermentations.

Adsorption↗

PubMed2002

Isolation and characterization of bacteriophages from fermenting sauerkraut.

This paper presents the first report of bacteriophage isolated from commercial vegetable fermentations. Nine phages were isolated from two 90-ton commercial sauerkraut fermentations. These phages were active against fermentation isolates and selected Leuconostoc mesenteroides and Lactobacillus plantarum strains, including a starter culture. Phages were characterized as members of the Siphoviridae and Myoviridae families. All Leuconostoc phages reported previously, primarily of dairy origin, belonged to the Siphoviridae family.

Bacteriophages↗

PubMed2002

Overexpression of peptidases in Lactococcus and evaluation of their release from leaky cells.

Walker and Klaenhammer (2001) developed a novel expression system in Lactococcus lactis that facilitated the release of beta-galactosidase (117 kDa monomer) without the need for secretion or export signals. The system is based on the controlled expression of integrated prophage holin and lysin cassettes via a lactococcal bacteriophage phi31 transcriptional activator (Tac31A) that resides on a high-copy plasmid. Approximately 85% of beta-galactosidase activity was detected in the supernatant of leaky lactococci without evidence of hindered growth, cell lysis, or membrane damage. The objective of this study was to determine if intracellular peptidases were externalized from leaky lactococci. Five L. lactis peptidases (PepA, PepC, PepN, PepO and PepXP) and two Lactobacillus helveticus peptidases (PepN and PepO) were cloned and overexpressed on two high-copy vectors. The lactococcal peptidases were also cloned into the high-copy vector that contained the Tac31A transcriptional activator to determine if they were externalized from the leaky prophage-containing L. lactis subsp. lactis strain NCK203. Two of the lactococcal peptidases (PepA and PepO) required an additional strong promoter (Lactobacillus paracasei P144) and optimized assay conditions to detect enzyme activity. Results showed different levels of enzymatic overexpression associated with the cellular fraction (2 to 250-fold increases in activity) and negligible amounts of activity present within the supernatant fraction (0 to 6% of total peptidase activity). The lactococcal phage-based protein release mechanism did not facilitate the externalization of the lactococcal peptidases investigated in this study.

Aminopeptidases↗

PubMed2001

Leaky Lactococcus cultures that externalize enzymes and antigens independently of culture lysis and secretion and export pathways.

A novel system that leaks beta-galactosidase (beta-gal) without a requirement for secretion or export signals was developed in Lactococcus lactis by controlled expression of integrated phage holin and lysin cassettes. The late promoter of the lytic lactococcal bacteriophage phi31 is an 888-bp fragment (P(15A10)) encoding the transcriptional activator. When a high-copy-number P(15A10)::lacZ.st fusion was introduced into L. lactis strains C10, ML8, NCK203, and R1/r1t, high levels of the resultant beta-gal activity were detected in the supernatant (approximately 85% of the total beta-gal activity for C10, ML8, and NCK203 and 45% for R1/r1t). Studies showed that the phenotype resulted from expression of Tac31A from the P(15A10) fragment, which activated a homologous late promoter in prophages harbored by the lactococcal strains. Despite the high levels of beta-gal obtained in the supernatant, the growth of the strains was not significantly affected, nor was there any evidence of severe membrane damage as determined by using propidium iodide or transmission electron microscopy. Integration of the holin-lysin cassette of phage r1t, under the control of the phage phi31 late promoter, into the host genome of MG1363 yielded a similar "leaky" phenotype, indicating that holin and lysin might play a critical role in the release of beta-gal into the medium. In addition to beta-gal, tetanus toxin fragment C was successfully delivered into the growth medium by this system. Interestingly, the X-prolyl dipeptidyl aminopeptidase PepXP (a dimer with a molecular mass of 176 kDa) was not delivered at significant levels outside the cell. These findings point toward the development of bacterial strains able to efficiently release relevant proteins and enzymes outside the cell in the absence of known secretion and export signals.

Antigens, Bacterial↗

PubMed2001

Analysis of the genetic switch and replication region of a P335-type bacteriophage with an obligate lytic lifestyle on Lactococcus lactis.

The DNA sequence of the replication module, part of the lysis module, and remnants of a lysogenic module from the lytic P335 species lactococcal bacteriophage phi31 was determined, and its regulatory elements were investigated. The identification of a characteristic genetic switch including two divergent promoters and two cognate repressor genes strongly indicates that phi31 was derived from a temperate bacteriophage. Regulation of the two early promoters was analyzed by primer extension and transcriptional promoter fusions to a lacLM reporter. The regulatory behavior of the promoter region differed significantly from the genetic responses of temperate Lactococcus lactis phages. The cro gene homologue regulates its own production and is an efficient repressor of cI gene expression. No detectable cI gene expression could be measured in the presence of cro. cI gene expression in the absence of cro exerted minor influences on the regulation of the two promoters within the genetic switch. Homology comparisons revealed a replication module which is most likely expressed from the promoter located upstream of the cro gene homologue. The replication module encoded genes with strong homology to helicases and primases found in several Streptococcus thermophilus phages. Downstream of the primase homologue, an AT-rich noncoding origin region was identified. The characteristics and location of this region and its ability to reduce the efficiency of plaquing of phi31 10(6)-fold when present at high copy number in trans provide evidence for identification of the phage origin of replication. Phage phi31 is an obligately lytic phage that was isolated from commercial dairy fermentation environments. Neither a phage attachment site nor an integrase gene, required to establish lysogeny, was identified, explaining its lytic lifestyle and suggesting its origin from a temperate phage ancestor. Several regions showing extensive DNA and protein homologies to different temperate phages of Lactococcus, Lactobacillus, and Streptococcus were also discovered, indicating the likely exchange of DNA cassettes through horizontal gene transfer in the dynamic ecological environment of dairy fermentations.

Amino Acid Sequence↗

PubMed2001

Identification and cloning of gusA, encoding a new beta-glucuronidase from Lactobacillus gasseri ADH.

The gusA gene, encoding a new beta-glucuronidase enzyme, has been cloned from Lactobacillus gasseri ADH. This is the first report of a beta-glucuronidase gene cloned from a bacterial source other than Escherichia coli. A plasmid library of L. gasseri chromosomal DNA was screened for complementation of an E. coli gus mutant. Two overlapping clones that restored beta-glucuronidase activity in the mutant strain were sequenced and revealed three complete and two partial open reading frames. The largest open reading frame, spanning 1,797 bp, encodes a 597-amino-acid protein that shows 39% identity to beta-glucuronidase (GusA) of E. coli K-12 (EC 3.2.1.31). The other two complete open reading frames, which are arranged to be separately transcribed, encode a putative bile salt hydrolase and a putative protein of unknown function with similarities to MerR-type regulatory proteins. Overexpression of GusA was achieved in a beta-glucuronidase-negative L. gasseri strain by expressing the gusA gene, subcloned onto a low-copy-number shuttle vector, from the strong Lactobacillus P6 promoter. GusA was also expressed in E. coli from a pET expression system. Preliminary characterization of the GusA protein from crude cell extracts revealed that the enzyme was active across an acidic pH range and a broad temperature range. An analysis of other lactobacilli identified beta-glucuronidase activity and gusA homologs in other L. gasseri isolates but not in other Lactobacillus species tested.

Amino Acid Sequence↗

PubMed2001

Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination.

An efficient method is described for the generation of site-specific chromosomal integrations in Lactobacillus acidophilus and Lactobacillus gasseri. The strategy is an adaptation of the lactococcal pORI system (K. Leenhouts, G. Venema, and J. Kok, Methods Cell Sci. 20:35-50, 1998) and relies on the simultaneous use of two plasmids. The functionality of the integration strategy was demonstated by the insertional inactivation of the Lactobacillus acidophilus NCFM lacL gene encoding beta-galactosidase and of the Lactobacillus gasseri ADH gusA gene encoding beta-glucuronidase.

Biotechnology↗

PubMed2001

Invited review: the scientific basis of Lactobacillus acidophilus NCFM functionality as a probiotic.

Lactobacillus acidophilus NCFM is a probiotic strain available in conventional foods (milk, yogurt, and toddler formula) and dietary supplements. Its commercial availability in the United States since the mid-1970s is predicated on its safety, its amenability to commercial manipulation, and its biochemical and physiological attributes presumed to be important to human probiotic functionality. The strain has been characterized in vitro, in animal studies, and in humans. NCFM is the progenitor of the strain being used for complete chromosome sequencing and therefore will be a cornerstone strain for understanding the relationship between genetics and probiotic functionality. Both phenotypic and genotypic techniques have verified its taxonomic status as a type A1 L. acidophilus strain. It adheres to Caco-2 and mucus-secreting HT-29 cell culture systems, produces antimicrobial compounds, and is amenable to genetic manipulation and directed DNA introduction. NCFM survives gastrointestinal tract transit in both healthy and diseased populations. NCFM inhibits aberrant crypt formation in mutagenized rats, indicative of activity that could decrease the risk of colon cancer. A blend of probiotic strains containing NCFM decreased the incidence of pediatric diarrhea. NCFM led to a significant decrease in levels of toxic amines in the blood of dialysis patients with small bowel bacterial overgrowth. At adequate daily feeding levels, NCFM may facilitate lactose digestion in lactose-intolerant subjects. Further validation of the probiotic properties of NCFM in humans and clarification of its mechanisms of probiotic action are needed to better understand the role this strain might play in promoting human health.

Anticarcinogenic Agents↗

PubMed2000

Use of the DNA sequence of variable regions of the 16S rRNA gene for rapid and accurate identification of bacteria in the Lactobacillus acidophilus complex.

The Lactobacillus acidophilus complex includes Lact. acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus gasseri and Lactobacillus johnsonii. The objective of this work was to develop a rapid and definitive DNA sequence-based identification system for unknown isolates of the Lact. acidophilus complex. A approximately = 500 bp region of the 16S rRNA gene, which contained the V1 and V2 variable regions, was amplified from the isolates by the polymerase chain reaction. The sequence of this region of the 16S rRNA gene from the type strains of the Lact. acidophilus complex was sufficiently variable to allow for clear differentiation amongst each of the strains. As an initial step in the characterization of potentially probiotic strains, this technique was successfully used to identify a variety of unknown human intestinal isolates. The approach described here represents a rapid and definitive method for the identification of Lact. acidophilus complex members.

Base Sequence↗

PubMed2000

Probiotic bacteria: today and tomorrow.

This paper provides an overview of the key issues raised during this symposium. Probiotic cultures have been associated historically with cultured milks and dairy products, from which there is substantial evidence for positive effects on human health and general well-being.

Animals↗

PubMed2000

An explosive antisense RNA strategy for inhibition of a lactococcal bacteriophage.

The coding regions of six putative open reading frames (ORFs) identified near the phage phi31 late promoter and the right cohesive end (cos) of lactococcal bacteriophage phi31 were used to develop antisense constructs to inhibit the proliferation of phage phi31. Two middle-expressed ORFs (ORF 1 and ORF 2) and four late-expressed ORFs (ORF 3 through ORF 6) were cloned individually between the strong Lactobacillus P6 promoter and the T7 terminator (T(T7)) to yield a series of antisense RNA transcripts. When expressed on a high-copy-number vector from a strong promoter, the constructs had no effect on the efficiency of plaquing (EOP) or the plaque size of phage phi31. To increase the ratio of antisense RNA to the targeted sense mRNA appearing during a phage infection, the antisense cassettes containing the late-expressed ORFs (ORF 3 through ORF 6) were subcloned to pTRK360, a low-copy-number vector containing the phage phi31 origin of replication, ori31. ori31 allows for explosive amplification of the low-copy-number vector upon phage infection, thereby increasing levels of antisense RNA transcripts later in the lytic cycle. In addition, the presence of ori31 also lowers the burst size of phage phi31 fourfold, resulting in fewer sense, target mRNAs being expressed from the phage genome. The combination of ori31 and P6::anti-ORF 4H::T(T7) resulted in a threefold decrease in the EOP of phage phi31 (EOP = 0.11 +/- 0.03 [mean +/- standard deviation]) compared to the presence of ori31 alone (EOP = 0.36). One-step growth curves showed that expression of anti-ORF 4H RNA decreased the percentage of successful centers of infection (75 to 80% for ori31 compared to 35 to 45% for ori31 plus anti-ORF 4H), with no further reduction in burst size. Growth curves performed in the presence of varying levels of phage phi31 showed that ori31 plus anti-ORF 4H offered significant protection to Lactococcus lactis, even at multiplicities of infection of 0.01 and 0.1. These results illustrate a successful application of an antisense strategy to inhibit phage replication in the wake of recent unsuccessful reports.

Bacteriophages↗

PubMed2000

Genetic analysis of chromosomal regions of Lactococcus lactis acquired by recombinant lytic phages.

Recombinant phages are generated when Lactococcus lactis subsp. lactis harboring plasmids encoding the abortive type (Abi) of phage resistance mechanisms is infected with small isometric phages belonging to the P335 species. These phage variants are likely to be an important source of virulent new phages that appear in dairy fermentations. They are distinguished from their progenitors by resistance to Abi defenses and by altered genome organization, including regions of L. lactis chromosomal DNA. The objective of this study was to characterize four recombinant variants that arose from infection of L. lactis NCK203 (Abi(+)) with phage phi31. HindIII restriction maps of the variants (phi31.1, phi31.2, phi31.7, and phi31.8) were generated, and these maps revealed the regions containing recombinant DNA. The recombinant region of phage phi31.1, the variant that occurred most frequently, was sequenced and revealed 7.8 kb of new DNA compared with the parent phage, phi31. This region contained numerous instances of homology with various lactococcal temperate phages, as well as homologues of the lambda recombination protein BET and Escherichia coli Holliday junction resolvase Rus, factors which may contribute to efficient recombination processes. A sequence analysis and phenotypic tests revealed a new origin of replication in the phi31.1 DNA, which replaced the phi31 origin. Three separate HindIII fragments, accounting for most of the recombinant region of phi31.1, were separately cloned into gram-positive suicide vector pTRK333 and transformed into NCK203. Chromosomal insertions of each plasmid prevented the appearance of different combinations of recombinant phages. The chromosomal insertions did not affect an inducible prophage present in NCK203. Our results demonstrated that recombinant phages can acquire DNA cassettes from different regions of the chromosome in order to overcome Abi defenses. Disruption of these regions by insertion can alter the types and diversity of new phages that appear during phage-host interactions.

Bacteriolysis↗

PubMed2000

Genetic modification of intestinal lactobacilli and bifidobacteria.

Lactobacilli and bifidobacteria are important members of the gastrointestinal microflora of man and animals. There is a substantial and growing body of evidence that these microbes provide benefits to the host in which they reside. Understanding the roles of these two groups of bacteria in the intestine continues to be a significant challenge. To this end, genetic characterisation and manipulation of intestinal lactobacilli and bifidobacteria is essential to define their contributions to the intestinal microflora, and to potentially exploit any beneficial or unique properties. This review will describe the tools and strategies currently available for the genetic manipulation of lactobacilli and bifidobacteria. Additionally, the ramifications and opportunities that may arise as a result of the genetic manipulation of probiotic lactobacilli and bifidobacteria will be addressed.

Animals↗

PubMed1999

Selection and design of probiotics.

Over the past 5 years the probiotic field has exploded with a number of new cultures, each purported to elicit a variety of benefits. Lists of functional characteristics and benefits, in vivo, are now commonplace to any presentation on probiotics. Scientifically established health claims remain among the highest priorities to companies who seek to establish solid health benefits that will promote their particular probiotic. The scientific community faces a greater challenge and must objectively seek cause and effect relationships for many potential and currently investigated probiotic species and strain combinations. Rational selection and design of probiotics remains an important challenge and will require a platform of basic information about the physiology and genetics of candidate strains relevant to their intestinal roles, functional activities, and interactions with other resident microflora. In this context, genetic characterization of probiotic cultures is essential to unequivocally define their contributions to the intestinal microbiota and ultimately identify the genotypes that control any unique and beneficial properties. Strain selection and differentiation, based on the genetic complement and programming of a candidate probiotic, then becomes feasible. Looking ahead, it will be vital to the development of this exploding field to correlate important characteristics in probiotics with known genotypes and regulatory controls that are likely to affect functionality and beneficial outcomes, in vivo.

Base Sequence↗

PubMed1999

Colicin V can be produced by lactic acid bacteria.

Colicin V is a small, proteinaceous bacterial toxin, produced by many strains of Escherichia coli and other members of the Enterobacteriaceae, that fits the definition of class II bacteriocins of Gram-positive bacteria. Export of colicin V is dependent on specific ABC (ATP-binding cassette) secretion proteins which recognize a double-glycine-type leader peptide on the immature colicin V bacteriocin. Replacement of the colicin V leader peptide by a signal peptide from the signal sequence-dependent bacteriocin divergicin A allowed expression of colicin V in lactic acid bacteria. This system may serve as a model for the heterologous expression of other small bacteriocins active against Gram-negative bacteria and other antibacterial peptides from lactic acid bacteria.

Bacteriocins↗

PubMed1999

Identification of the pH-inducible, proton-translocating F1F0-ATPase (atpBEFHAGDC) operon of Lactobacillus acidophilus by differential display: gene structure, cloning and characterization.

The influence of low pH on inducible gene expression in Lactobacillus acidophilus was investigated by the use of differential display. Logarithmic phase cultures were exposed to pH 3.5 for various intervals, and RNA was isolated and reverse transcribed. The resultant cDNAs were subjected to PCR and the products were resolved by electrophoresis. Several cDNA products were induced after exposure to pH 3.5. One of these products, a 0.7 kb fragment, showed sequence similarity to bacterial atpBEF genes of the atp operon, whose genes encode the various subunits of the F1F0-ATPase. With the 0.7 kb differential display product as a probe, hybridizations with total RNA from untreated and acid-treated L. acidophilus verified the acid inducibility of this operon. The increase in atp mRNA induced by low pH was accompanied by an increase in the activity of the enzyme in membrane extracts. The full-length atp operon was sequenced, and its genes were in the order of atpBEFHAGDC, coding for the a, c, b, delta, alpha, gamma, beta and epsilon subunits respectively. The operon contained no i gene, but was preceded by a 122 bp intergenic space, which contained putative extended -10 and -35 promoter regions. Primer extension analysis of RNA from cultures that were shifted from pH 5.6 to pH 3. 5, and held for 0, 30 or 45 min, revealed that the transcriptional start site did not change position as a function of culture pH or time after exposure to pH 3.5. The primary structure and genetic organization indicated that the H+-ATPase of L. acidophilus is a typical F1F0-type ATPase. The similarity to streptococcal ATPases and the acid inducibility of this operon suggest that it may function in the ATP-dependent extrusion of protons and maintenance of cytoplasmic pH. Finally, the use of differential display RT-PCR was an effective approach to identify genes in L. acidophilus induced by an environmental stimulus.

Amino Acid Sequence↗