Lactococcal 936-species phage attachment to surface of Lactococcus lactis

The interactions of the 936-species phages sk1, jj50, and 64 with the cell surface of Lactococcus lactis LM0230 were analyzed. Cell envelopes (walls + plasma membrane), cell wall, or plasma membrane from L. lactis ssp. lactis LM0230 each inactivated the phages in vitro. However, other 936-species phages kh and P008, which do not infect strain LM0230, were not inactivated by any of the subcellular fractions. Treating cell walls or plasma membrane with the cell wall hydrolase mutanolysin eliminated inactivation of phage sk1. This suggested that intact cell wall fragments were required for inactivation. A role for plasma membrane in phage sk1 inactivation was further investigated. Boiling, washing in 2 M KCl, 8 M urea, or 0.1 M Na(2)CO(3)/pH 11, or treating the plasma membrane with proteases did not reduce adsorption or inactivation of phage. Adding lipoteichoic acid or antibodies to lipoteichoic acid did not reduce inactivation of phage in a mixture with membrane, suggesting that lipoteichoic acid was not involved. Inactivation by envelopes or cell wall correlated with ejection of DNA from the phage sk1 capsid. Although calcium is required for plaque formation, it was not required for adsorption, inactivation, or ejection of phage DNA by envelopes or cell wall. The results suggest that at least for phages sk1, jj50, and 64, adsorption and phage DNA injection into the host does not require a host membrane protein or lipoteichoic acid, and that cell wall components are sufficient for these initial steps of phage infection.     

Purification and characterization of a cell wall proteinase from Streptococcus lactis NCDO 763

A proteinase was purified from a cell wall extract of a culture of Streptococcus lactis NCDO 763 grown in skim milk. Being active at a low pH (at pH 4.8 on haemoglobin and pH 6.0-6.5 on casein) and completely inhibited by diisopropylfluorophosphate, it was considered to be a serine proteinase partly inhibited by EDTA; the mol. wt was approximately 80,000.     

Molecular architecture of the lactococcal cell surface as it relates to important industrial properties

From an industrial perspective there are a number of important properties of lactococci that are either carried on, or are controlled and regulated by, the cell surface e.g. phage adsorption, exopolysaccharide production, cell aggregation/sedimentation, cell autolysis and the translocation of foreign DNA. Lactococcus lactis subsp. cremoris strain E8 and its spontaneous phage-resistant variant strain 398 were used as a model system to study the molecular architecture of the lactococcal cell surface and to identify the cell wall components that are involved in phage adsorption. Genetically, the two strains were found to be very closely related as determined by the comparison of chromosomal DNA and plasmid profiles.

Two major cell wall components of both strains were the accessory polysaccharides and peptidoglycan, the latter containing, Asp, Glu, Lys and Ala in molar ratio of 1:1:1:2, respectively. There were, however, significant quantitative differences in the levels of monosaccharide in accessory polysaccharide of the two strains; the parent strain contained 15-fold more galactose and 3-fold more glucosamine.

In order to identify the components of the cell wall involved in phage adsorption, the cell walls from the parent strain (strain E8) were subjected to various chemical and enzymatical treatments before testing their ability to adsorb different phages. The result indicated that the essential component of the phage receptor in the cell wall of Lc. lactis subsp. cremoris strain E8 was carbohydrate in nature and was covalently linked to the peptidoglycans. Lectin binding experiments revealed that galactose and glucosamine were required for the adsorption of phages. An electron microscopic study of the interaction between the phages and their host showed that phages adsorb to a limited number of receptor sites which were distributed over the entire cell envelope.     

Effectiveness of thermal treatments and biocides in the inactivation of Argentinian Lactococcus lactis phages

The thermal and chemical resistance levels of four autochthonal bacteriophages of Lactococcus lactis subsp. lactis, isolated from cheese processes, was investigated. The times required to obtain 99% inactivation of phages (T99) at 63 and 72 degrees C in three suspension media (M17 broth, reconstituted commercial nonfat skim milk, and Tris magnesium gelatin buffer) were determined. Thermal resistance was dependent on the phage studied, and the results of this study demonstrate that pasteurization treatments used in dairy industries may leave viable viral particles in milk. It was possible to determine that M17 broth was generally the least protective medium, while phosphate buffer was the most protective one. Peracetic acid (0.15%, vol/vol) was the most effective viricidal agent, with exposures of 5 min being sufficient to inactivate high-titer phage suspensions (>10(6) PFU/ml). To achieve total inactivation (<10 PFU/ml) of viral suspensions, sodium hypochlorite was effective at 100 ppm for only two phages, while the other two phages needed concentrations of 200 and 300 ppm. Ethanol at concentrations of 100 and 75% proved to be very efficient in inactivating phages, but isopropanol was not effective against them.     

Growth stimulation of a proteinase positive Lactococcus lactis strain by a proteinase negative Lactococcus lactis strain

Lactococcus lactis AMP15/pAMP31(D471R) is a proteinase negative, lactose negative strain with a modified oligopeptide transport system, and potential as a debittering agent due to its efficient utilization of hydrophobic peptides. Five wild L. lactis strains of dairy origin, which produced cheeses of high flavour quality, were cocultured with L. lactis AMP15/pAMP31(D471R) in an attempt to select adequate combinations of strains for use as defined cheese starters with potential debittering ability. Four of these strains, L. lactis B6, K16, M21 and P21, inhibited growth of L. lactis AMP15/pAMP31(D471R) at a level of 10(6) to 10(7) cfu mL(-1) after 24 h of incubation, even though production of bacteriocin-like compounds could only be proven for L. lactis M21. When L. lactis AMP15/pAMP31(D471R) was cocultured with the fifth strain, L. lactis N22, its growth was significantly (P<0.001) inhibited whereas growth of L. lactis N22 was significantly stimulated. The nature of the interaction was studied and it was established that L. lactis N22 is auxotrophic for folate, a compound produced and excreted by L. lactis AMP15/pAMP31(D471R).     

Characterization of four new Lactococcus lactis bacteriophages isolated from dahi whey

Four lytic phages of Lactococcus lactis ssp. diacetylactis isolated from indigenous dahi whey were examined for their stability, growth characteristics and morphology. All these phages were partially inactivated by CHCl₃ , remained stable at 40°C and were partially inactivated at pH 3. There was a marked difference among these phages with respect to latent period, rise period and burst size. All phages belonged to Bradley's group B.     

Characterisation of technologically proficient wild Lactococcus lactis strains resistant to phage infection

The aim of this work was to establish whether Lactococcus lactis strains isolated from spontaneous dairy fermentations exhibited useful milk-processing capabilities and resistance to bacteriophage infection in order to be used as components in starter formulations. The 33 out of 100 isolates of L. lactis, originated from farmhouse cheeses, were found to be resistant to a collection of 34 phages belonging to the c2 and 936 groups. Six of the isolates were discarded as potential starters because they were lysogenic and other five because they produced tyramine. Plasmid and chromosomal profiles of the 22 remaining isolates allowed their classification into 16 different strains. All of these were good lactic acid producers from lactose, moderately proteolytic and, in eight cases, diacetyl production from citrate was observed. The mechanism(s) leading to the phenotype of phage resistance was identified for all the strains used in this study. Inhibition of adsorption was the most frequent one, although genetic determinants for some abortive infection systems were also detected (abiB, abiG and abiI). Frequently, more than one mechanism was present in the same strain. One of the strains, L. lactis IPLA542, was selected as a model starter for pilot fermentations. It clotted milk normally both in the absence and in the presence of phage at concentrations that completely abolished the process when promoted by a phage-susceptible strain.     

Determination of metabolic plasmids and their effects on the growth of Lactococcus lactis ssp. lactis MN24

Mutational analyses revealed the 21.5 kb plasmid-encoded lactose fermentation and proteolytic activity properties in Lactococcus lactis ssp. lactis MN24. Reductions in maximum specific growth rate and population density of the 21.5 kb plasmid-cured mutant of MN24 confirmed the data obtained by mutation tests. Plasmid curing, polymerase chain reaction and DNA sequence analyses data showed that the lacticin 481 operon was located on 22.4 kb plasmid. The phage resistance system in strain MN24 was identified as an adsorption inhibition type and chromosomally encoded via phage–host interaction tests and mutation analyses.     

vga Mutants of Kluyveromyces lactis show cell integrity defects

We studied the cell wall alterations that occur in mutants of Kluyveromyces lactis impaired in glycosylation. The mutants belong to four complementation groups named vga1 to vga4 (vanadate glycosylation affected), characterized by sodium orthovanadate resistance and alteration of the glycosylation profile of native invertase. A drastic reduction of the alkali-soluble fraction of the beta-D-glucan was observed in vga1, vga2 and vga3 cells, accompanied by an increase in the chitin content of the cell wall. In vga4 cells, both beta-D-glucan fractions (alkali-soluble and alkali-insoluble) were reduced to about half of the corresponding wild-type value but the chitin content was normal. A protein related to Fks1p, the catalytic subunit of the major 1,3-beta-D-glucan synthase of S. cerevisiae, was detected in K. lactis. The amount of this Fks1p-like protein increased 7-10 times in vga1, vga2 and vga3 mutants as compared to wild-type cells; the same strains released significant amounts of beta-D-glucan in the culture supernatant. These mutations also resulted in abnormally thick cell walls with conspicuous irregularities in the structure, as revealed by electron microscopy and by an altered resistance to Zymolyase. The observed high responsiveness of cell wall phenotypes to alterations of glycosylation make K. lactis an attractive system for studying the interconnections between these processes.     

Screening for natural defence mechanisms of Lactococcus lactis strains isolated from traditional starter cultures

Three different bacterial defence mechanisms were identified in the seventeen Lactococcus lactis isolates from starter cultures in three Slovenian dairy plants. Isolates MB18, KR7, PT4, PT13 and PT19 inhibited phage adsorption by means of exopolysaccharides production. The most extensive polysaccharides production was detected in PT19 isolate, which was susceptible only to phage ΦPT19. Eight isolates exhibited nuclease activity, and seven of them were susceptible up to four phages out of thirteen from our collection. Eight isolates possessed the abiB gene, fourteen isolates abiH, two isolates abiJ and one isolate abiQ. Isolates PT27 and PT28 possessed AbiB, AbiH and AbiJ mechanisms as well as inhibition of phage adsorption. Isolate MB18, which was susceptible to one phage only, possessed the abiQ gene, nuclease activity and ability to prevent adsorption of most phages. Isolates PT67 and PT70, possessing only AbiH mechanism, were susceptible to only two phages.     

Growth associated exopolysaccharide expression in Lactococcus lactis subspecies cremoris Ropy352

A natural lactococcal isolate, Lactococcus lactis ssp. cremoris Ropy352, has been previously shown to express two phenotypically distinct exopolysaccharides (ropy and mucoid). This natural isolate was cultured on various media to explore the carbon requirements for exopolysaccharide expression. Ropy exopolysaccharide expression was optimal when grown in defined media rather than on M17-based media. Ropy352 was examined for inducible lysogenic phages. No lytic burst was observed in Ropy352 with ultraviolet light or mitomycin C for phage induction. The sugar compositions of the two phenotypically distinct exopolysaccharides were determined. The ropy exopolysaccharide is composed of galactose and glucose in the molar percents of 42 and 58%, respectively. The mucoid exopolysaccharide is composed of galactose, glucose, and mannose in the molar percents of 58, 29, and 13%, respectively. Mutational analysis revealed that mutations impairing ropy exopolysaccharide expression but not affecting mucoid exopolysaccharide expression could be isolated.     

一株Kluyveromyces lactis酵母胞内乳糖酶性质的研究

源自乳酸克鲁维酵母的β-半乳糖苷酶为胞内酶,其具有乳糖水解能力和半乳糖苷的转移作用。本实验运用单因素试验方法研究乳酸克鲁维酵母乳糖酶的性质。结果表明该酶在pH值为6.0~7.0和37℃~48℃间比较稳定,酶作用最适pH值在6.5,最适反应温度为43℃,Mn2+、Mg2+等对酶有明显的激活作用,而Zn2+、Cu2+等对酶活有抑制作用。该酶以ONPG底物的米氏常数为4.186mmol/L。     

Viability of probiotic micro-organisms (Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp. lactis Bb-12) in ice cream

The purpose of this research was to determine the survival of two probiotic micro-organisms in ice creams (4% fat). The micro-organisms were Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp . lactis Bb-12 . To meet this objective, an ice cream mixture was formulated and subjected to three treatments. Treatment 1 was inoculated with L. acidophilus , treatment 2 with B. lactis and the third treatment was inoculated with a mixture of both bacteria inoculated in 1 : 1 proportions. The inoculation was with 4% culture for each treatment. The final products were stored at −25°C for 60 days. The ice cream inoculated with L. acidophilus had a final concentration of 2 × 10 ⁶ cfu/g and the survival rate was 87%. The treatment inoculated with B. lactis had a final concentration of 9 × 10 ⁶ cfu/g, with a logarithmic decrease of 10%. When both micro-organisms were inoculated together, the survival rate was 86%.     

Bacteriophage resistance in Lactococcus lactis ssp. lactis using antisense ribonucleic acid.

Antisense RNA against a conserved bacteriophage gene when expressed in a Lactococcus lactis ssp. lactis strain renders it resistant to bacteriophage infection. Two open reading frames have been identified in a L. lactis ssp. lactis bacteriophage that are conserved in a majority of isolates. They code for an 18-kDa (designated GP18C) protein and a 24-kDa (GP24C) protein, respectively, which are arranged along with previously identified open reading frames in a tandem motif similar to other bacteriophages. The presence of gp18C and gp24C in a number of bacteriophage isolates was confirmed by polymerase chain reaction using primers specific for these regions. Plasmids bearing various fragments of gp18C, gp24C, or both were constructed such that the respective open reading frames were positioned in the antisense direction relative to the Lactococcus lactis ssp. cremoris Wg2 promoter, p59. These antisense RNA-producing vectors inhibited the efficiency of plaquing of L. lactis ssp. lactis bacteriophage phi 7-9 up to 50%; the resulting plaques were extremely small and irregular in shape. The replication of the bacteriophage was severely inhibited, and the total number decreased over the first 3 h during infection in strains expressing antisense RNA compared with the host strain alone, in which the bacteriophage number increased 10(4)-fold.     

Mode of action of lactococcin R produced by Lactococcus lactis R

We investigated the mode of action and factors affecting adsorption of lactoccocin R produced by Lactococcus lactis R. It was found that lactococcin R adsorbed to all Gram-positive but not to the Gram-negative bacteria tested and its adsorption was dependent on pH. It was observed that the binding of lactococcin R was prevented by anions of several salts (Cl-, PO4(-3)) and lipoteichoic acid. Pretreatments of sensitive cells and cell walls with detergents, organic solvents or enzymes did not reduce subsequent binding of lactococcin R. However, treatment of cell wall preparations with methanol:chloroform and hot 20% trichloroacetic acid (TCA) caused such walls to lose their ability to adsorb lactococcin R. Sensitive cells treated with lactococcin R lost high amounts of intracellular K+ ions, UV-absorbing materials and became more permeable to o-nitrophenol-beta-D-glactopyranoside (ONPG). In addition, different lactococcin R concentrations (0-2560 AU/mL) decreased the colony counts of Listeria monocytogenes by 99% and also a reduction in the absorbance values. These results show that the mode of action of lactococcin R is bactericidal rather than bacteriostatic.     

The ubiquitin-encoding genes of Kluyveromyces lactis

The ubiquitin encoding genes of Kluyveromyces lactis were cloned. Three genes, KlUBI1, KlUBI3 and KlUBI4, were found in this yeast, while in Saccharomyces cerevisiae there are four genes, UBI1, -2, -3 and -4. The UBI1/UBI2 duplication is thus absent from the K. lactis genome. General structural features of ubiquitin genes were very similar in these two species (presence of an intron in KlUBI1, fusion to ribosomal protein genes in KlUBI1 and KlUBI3, spacer-less polyubiquitin repeats in KlUBI4). Disruption or deletion of K. lactis ubiquitin genes showed that: (a) disruption of KlUBI1 was lethal (in S. cerevisiae, ubi1/ubi2 double deletion is lethal); (b) KlUBI3 is also an essential gene for cell growth; (c) deletion of KlUBI4 led to an increased sensitivity to high temperature, similar to the ubi4 mutation in S. cerevisiae, but, in contrast to the latter, the klubi4 mutant was not sensitive to carbon or nitrogen source starvation. The syntenic relationship of ubiquitin loci between K. lactis and S. cerevisiae genomes is also described.     

外源添加亮氨酸提高乳酸乳球菌酸胁迫抗性

在乳酸乳球菌生长过程中外源添加浓度为10mmol/L的亮氨酸,可有效提高乳酸乳球菌在酸胁迫环境下的酸耐受性。在酸性环境中(pH5.0),添加亮氨酸的菌株的生物量为对照菌株的1.24倍;经过酸胁迫(pH4.0)5h后,添加亮氨酸菌株的存活率是对照菌株的28.5倍。进一步的研究表明,亮氨酸的添加可提高胞内NH4+浓度,有效的维持酸胁迫环境下胞内pH(pHin)的稳定,并有效维持乳酸脱氢酶(LDH)的活性,从而有效提高了乳酸乳球菌对酸胁迫的抵御能力。     

The proteolytic system of the yeast Kluyveromyces lactis.

Major proteolytic activities were characterized in the yeast K. lactis NRRL 1118, grown in chemostat cultures. This yeast expressed proteolytic activities similar to those found in S. cerevisiae. This fact was particularly evident in the case of proteases such as PrA, PrB and CpY with regard to substrate specificity, activation at pH 5. 0 and inhibition patterns. The presence of a CpS activity could not be detected in either fresh or activated cell-free extracts by using the dipeptide N-Cbz-Gly-Leu, even in the presence of Zn(+2). On the other hand, K. lactis exhibits at least two major intracellular Ap activities different from those reported in other yeasts, and these seem to be carried out by closely related proteins. These activities corresponded to molecular masses of about 60 kDa, close pI values, and a similar behaviour in non-denaturing polyacrylamide electrophoresis. Both activities were enhanced by Co(+2) and inhibited by EDTA. Among different aminoacyl-p-NAs, they preferentially hydrolysed Lys-p-NA. No increase of Ap activity was obtained by incubation of extracts at acid pH. The maximum PrA and PrB activities detected in N-limited cultures were six-fold higher than those expressed under C- or P-limitation. The effect of culture conditions on the Cp and Ap expression was much less pronounced in comparison with PrA and PrB activities, Ap levels even being slightly higher in C-limited cells. This fact suggests that hydrolysis of protein to peptides might be the limiting step in the pathway of general protein degradation in the vacuole.     

Isolation of halotolerant Lactococcus lactis subsp. lactis from intestinal tract of coastal fish

We isolated lactic acid bacteria from the intestinal tract of the pufferfish Takifugu niphobles caught in Shimoda, Shizuoka, Japan by using MRS broth prepared with 50% seawater. Additional screening was carried out using phenotypic tests such as Gram staining, cell morphology, catalase, oxidase and fermentation of glucose. Subsequently 227 isolates screened by the phenotypic tests were subjected to species-specific PCR for Lactococcus lactis, resulting in four positive isolates. The 16S rRNA gene sequences from three isolates were highly similar to that of L. lactis subsp. lactis (DNA database accession number M58837), while that of one isolate was identical to that of Leuconostoc mesenteroides (AB023246). These isolates were characterized by API 50 CH for carbohydrate fermentation and other phenotypic criteria for salt tolerance, and the characteristics were compared with those of L. lactis subsp. lactis from a cheese starter culture. The carbohydrate fermentation profiles of these isolates were characteristic of L. lactis subsp. lactis strains, whereas the tolerance of these isolates to salt was higher than that of L. lactis subsp. lactis from the cheese starter culture: the new L. lactis isolates showed high salt tolerance in MRS-agar plates containing 200% seawater or 6% sodium chloride. This is the first report of the isolation of halotolerant strains of L. lactis subsp. lactis from a marine environment.     

Surface physicochemical analysis of natural Lactococcus lactis strains reveals the existence of hydrophobic and low charged strains with altered adhesive properties

The cell surface physicochemical properties of 50 Lactococcus lactis strains of different subspecies and isolated from different origins (dairy, vegetal and animal) were examined. Cell surface hydrophobicity and Lewis acid-base properties were evaluated by affinity measurements to solvents in a partitioning test, while the global electrical charge of the cells was assessed by micro-electrophoresis using a laser zeta-meter. A global multivariate analysis of the results revealed a high natural diversity of L. lactis cell surface properties. While 52% of the strains present a hydrophilic and electronegative cell wall surface, a group of strikingly hydrophobic strains (12% of the strains) and a group of strains with unusual low charged surface (18%) were identified. Adhesion on polystyrene microtitre plates was evaluated for twelve strains selected from the multivariate analysis as representatives of the various observed cell wall surface physicochemical patterns. A significant correlation between adhesion, hydrophobicity and low electronegativity was observed when adhesion was performed in a low ionic strength suspending medium. The most adhesive strains were hydrophobic or low charged. The presence of repulsive electrostatic interactions led to a decrease in adhesion of the most negatively charged hydrophilic strains. The present study highlights the diversity of L. lactis cell surface physicochemical properties, diversity that could not be connected to the origin or to the subspecies of the strains.