Purification and properties of the alpha-acetolactate decarboxylase from Lactococcus lactis subsp. lactis NCDO 2118

The authors report the case of a very large deterged aortic valve ring abscess detected at long-term after infective endocarditis during investigation of symptomatic consequent aortic regurgitation. The different imaging methods of diagnostic import are reviewed with special emphasis on the role of transoesophageal echocardiography during infective endocarditis.     

Effect of citrate on growth of Lactococcus lactis subsp. lactis in milk

The effect of citrate on the growth of Lactococcus lactis subsp. lactis var. diacetylactis in milk has been investigated. Five strains of Lactococcus lactis subsp. lactis var. diacetylactis were compared to their citrate-negative variants, which lack the plasmid coding for citrate permease. In most cases, acidification kinetics and the final bacterial concentration of pure cultures of parental and variant strains did not differ significantly. Co-cultures of parental and variant strains, however, systematically tended towards the predominance of parental strains. Citrate metabolism is responsible for this change, since the predominance of citrate-positive strains was not observed in the absence of citrate. Continuous culture in milk enabled the difference in growth rates between the parental strain Lactococcus lactis subsp. lactis var. diacetylactis CDI1 and its citrate-negative variant to be quantified by following changes in the populations of the two co-cultured strains. At 26 degrees C, the growth rate of the parental strain was 7% higher than that of its citrate-negative variant. These results show that citrate metabolism slightly stimulates the growth of lactococci in milk.     

Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts

Knowledge of the basic economic factors underlying managed mental health care directly impacts the clinical practitioners' ability to make constructive changes in the system. To aid understanding this article introduces the managed care marketplace model, the interactive relationship between medical necessity and patient co-payment, and demand management economics. The author encourages practitioners to develop strategies to overcome specific economic obstacles that prevent the promotion of group psychotherapy.     

Homing of a group II intron from Lactococcus lactis subsp. lactis ML3

Ll.ltrB is a functional group II intron located within a gene (ltrB) encoding a conjugative relaxase essential for transfer of the lactococcal element pRSO1. In this work, the Ll.ltrB intron was shown to be an independent mobile element capable of inserting into an intronless allele of the ltrB gene. Ll.ltrB was not observed to insert into a deletion derivative of the ltrB gene in which the intron splice site was removed. In contrast, a second vector containing a 271-nucleotide segment of ltrB spanning the Ll.ltrB splice site was shown to be a proficient recipient of intron insertion. Efficient homing was observed in the absence of a functional host homologous recombination system. This work demonstrates that the Ll.ltrB intron is a novel site-specific mobile element in lactococci and that group II intron self-transfer is a mechanism for intron dissemination among bacteria.     

A deficiency in aspartate biosynthesis in Lactococcus lactis subsp. lactis C2 causes slow milk coagulation

A mutant of fast milk-coagulating (Fmc+) Lactococcus lactis subsp. lactis C2, designated L. lactis KB4, was identified. Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc-) phenotype. When the amino acid requirements of L. lactis C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid. This aspartic acid requirement could also be met by aspartate-containing peptides. The addition of aspartic acid to milk restored the Fmc+ phenotype of KB4. KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide. The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis.     

Purification and characterization of aminopeptidase P from Lactococcus lactis subsp. cremoris

BACKGROUND: Neutrophils, platelets, and cytokines are thought to play a pivotal role in the pathogenesis of endotoxin-induced lung injury which resembles features of acute respiratory distress syndrome (ARDS). For initiation of this pathological process, neutrophils and platelets are activated and adhere to pulmonary endothelium. Nitric oxide (NO) inhibits adhesion and activation of these cells and decreases the cytokine level in bronchoalveolar lavage (BAL) fluid obtained from patients with ARDS. Limited data are available on the effect of NO treatment before and after endotoxin on the development and advance of ARDS. The aim of the current study was to determine whether NO inhalation prevents acute lung injury. METHODS: Thirty-two male anaesthetized rabbits were randomly assigned to receive one of four treatments (n = 8 each); Group S-N received saline with nitrogen (N2), Group S-NO received saline infusion with NO (20 p.p.m.) inhalation, Group E-N received an infusion of Escherichia coli endotoxin 100 micrograms/ kg over 60 min with inhalation of N2, and Group E-NO received endotoxin with NO (20 p.p.m.) inhalation. The lungs of the rabbits were ventilated with 40% oxygen until 6 h after the start of endotoxin or saline administration. Haemodynamics and PaO2 were recorded during the ventilation period. After observation, the lung wet-to dry-(W/D) weight ratio, lung mechanics, and cell fraction, activated complements, cytokines, arachidonic acid metabolites, and albumin concentrations in the BAL fluid were measured and analysed. Light microscopic findings were compared among the four groups. RESULTS: Pulmonary hypertension and deterioration of oxygenation by endotoxin were less pronounced in rabbits receiving NO. The lung compliance after endotoxin was similar in Groups E-NO and E-N. The W/D weight ratio and neutrophils and albumin concentrations in the BAL fluid increased in Groups E-NO and E-N. The BAL fluid concentrations of interleukin-8, thromboxane A2, and prostacyclin were similar in the two endotoxin-treated groups. Endotoxin caused extensive morphologic lung damage regardless of NO inhalation. CONCLUSIONS: The increase in pulmonary arterial pressure and deterioration of oxygenation were less in endotoxin-exposed rabbits receiving NO inhalation compared with those receiving N2. Accumulation of neutrophils and platelets in the lung, morphological lung damage, and the release of cytokines and prostanoids were observed in the E-NO group. However, we are unable to extrapolate these results directly to the human clinical setting because of the short observation period, the use of only one dose of NO, and the species difference.     

Studies on purification and some properties of nisin from Lactococcus lactis subsp. lactis Al2

Protoporphyria is a genetic disorder in which patients may develop severe protoporphyrin-induced liver damage and require transplantation. Because unique problems occur in the perioperative period and because excess production of protoporphyrin by the bone marrow continues after liver transplantation, the efficacy of this procedure for protoporphyric liver disease is uncertain. We present follow-up of nine patients who underwent liver transplantation. Two patients died within 2 months of transplantation, one from complications of abdominal bleeding and the other from sepsis after bowel perforations. The remaining seven patients had follow-up at 14 months to 8 years after transplantation (mean, 3.8 years). Two of the seven had suffered skin burns from exposure to operating room lights, which healed without scarring. Three had axonal neuropathies in the postoperative period requiring prolonged mechanical ventilation, and motor defects persisted in two. Five patients had normal liver chemistries at follow-up (mean, 3.5 years), with liver biopsy results normal or showing mild portal triad abnormalities, but erythrocyte protoporphyrin levels remained significantly elevated (1,765 +/- 365 mcg/dL; normal, < 65). The other two patients, both of whom had rejection, cytomegalovirus infection, and biliary tract obstruction requiring endoscopic therapy, had a recurrence of protoporphyric liver disease as indicated by liver biopsy features. One died 5 years after transplantation from complications of the liver disease. The other was stable 3.3 years after transplantation and was being monitored for possible retransplantation. Thus, liver transplantation can be performed successfully in patients with protoporphyric liver disease, with intermediate survival rates comparable to the general transplant population. However, disease may recur in the graft, particularly if there are complications that cause cholestasis.     

Characterization of LlaCI, a new restriction-modification system from Lactococcus lactis subsp. cremoris W15

The genes encoding the restriction-modification (R/M) system LlaCI have been found on the naturally occurring 7.0 kb plasmid pAW153 in L. lactis subsp. cremoris W15. The R/M system was isolated on a chloramphenicol resistant derivative of the wild type plasmid (pAW153cat). Plasmid pAW153cat and a 2.4 kb HincII-SphI fragment cloned into a high- and a low-copy vector conferred decreased sensitivity in L. lactis LM2301 and L. lactis SMQ86 against small isometric-headed phages of the 936 or P335 species, respectively. Increased plasmid copy number enhanced the level of phage restriction. Sequencing the 2.4 kb HincII-SphI fragment revealed two open reading frames arranged convergently with a 94 bp separation. IlaCIM showed 66% identity to hindIIIM, and IlaCIR showed 45% identity to hindIIIR. The organization of the LlaCI operon differs from the HindIII operon, where the endonuclease and methylase genes overlap and are transcribed in the same direction. The LlaCI methylase is predicted to be 296 amino acids long, with 63% identity to the HindIII methylase, while the LlaCI endonuclease is predicted to consist of 324 or 332 amino acids, depending on the position of the start codon. It shows 24% identity to the HindIII endonuclease.     

Isolation, cloning and characterisation of the abiI gene from Lactococcus lactis subsp. lactis M138 encoding abortive phage infection

Morphological and morphometric features of the cornea of 13 species of primates have been studied in order to determine possible morphological differences between them. The existence of relationships between different morphometric corneal variables was also examined to establish which variables best defined and characterized the cornea. The present aim is to determine which primate cornea resembles that of the human being most with a view to possible future clinical and experimental studies. The results obtained revealed that all the cornea under study presented similar morphological features. The relationship between total corneal thickness and corneal epithelial thickness was determined as well as the relationship between epithelial thickness, the number of epithelial layers and the number of epithelial cells. However, the morphological pattern of Bowman's membrane and corneal endothelium differed in the species studied. Finally, the study indicates that the chimpanzee and the gorilla are the species with a corneal morphometry which is closest to that of the human cornea.     

Lysis of Lactococcus lactis subsp. cremoris SK110 and its nisin-immune transconjugant in relation to flavor development in cheese

To develop a nisin-producing cheese starter, Lactococcus lactis subsp. cremoris SK110 was conjugated with transposon Tn5276-NI, which codes for nisin immunity but not for nisin production. Cheese made with transconjugant SK110::Tn5276-NI as the starter was bitter. The muropeptide of the transconjugant contained a significantly greater amount of tetrapeptides than the muropeptide of strain SK110, which could have decreased the susceptibility of the cells to lysis and thereby the release of intracellular debittering enzymes.     

Influence of intracellular pH on light emission from a luxA/B derivative of Lactococcus lactis subsp. diacetylactis

High levels of constitutive aldehyde-dependent light emission were obtained from nongrowing cells of Lactococcus lactis subsp. diacetylactis F712 transformed with IuxA/B when they were suspended in buffered solutions. Inductions of light emission was time-dependent and was not due to growth, synthesis of luciferase or stimulation of metabolism by fermentable carbohydrate. The major factor controlling light emission in such cells appears to be the intracellular pH value. Experiments with ionophores indicated that a transmembrane pH gradient was not essential for light emission.     

Specificity of milk peptide utilization by Lactococcus lactis

To study the substrate specificity of the oligopeptide transport system of Lactococcus lactis for its natural substrates, the growth of L. lactis MG1363 was studied in a chemically defined medium containing milk peptides or a tryptic digest of alpha s2-casein as the source of amino acids. Peptides were separated into acidic, neutral, and basic pools by solid-phase extraction or by cation-exchange liquid chromatogrpaphy. Their ability to sustain growth and the time course of their utilization demonstrated the preferential use of hydrophobic basic peptides with molecular masses ranging between 600 and 1,100 Da by L. lactis MG1363 and the inability to use large, acidic peptides. These peptide utilization preferences reflect the substrate specificity of the oligopeptide transport system of the strain, since no significant cell lysis was inferred. Considering the free amino acid content of milk and these findings on peptide utilization, it was demonstrated that the cessation of growth of L. lactis MG1363 in milk was due to deprivation of leucine and methionine.     

Autolysis of Lactococcus lactis is influenced by proteolysis

The autolysin AcmA of Lactococcus lactis was shown to be degraded by the extracellular lactococcal proteinase PrtP. Autolysis, as evidenced by reduction in optical density of a stationary-phase culture and concomitant release of intracellular proteins, was greatly reduced when L. lactis MG1363 cells expressed the cell wall-anchored lactococcal proteinase PrtP of the PI-type caseinolytic specificity (PI). On the other hand, lactococcal strains that did not produce the proteinase showed a high level of autolysis, which was also observed when the cells produced the secreted form of PI or a cell wall-anchored proteinase with PIII-type specificity. Autolysis was also increased when MG1363 expressed the cell wall-anchored hybrid PI/PIII-type proteinase PIac. Zymographic analysis of AcmA activity during stationary phase showed that AcmA was quickly degraded by PI and much more slowly by PrtP proteinases with PIII-type and intermediate specificities. Autolysis of L. lactis by AcmA was influenced by the specificity, amount, and location of the lactococcal proteinase. No autolysis was observed when the various proteinases were expressed in an L. lactis acmA deletion mutant, indicating that PrtP itself did not cause lysis of cells. The chain length of a strain was significantly shortened when the strain expressed a cell wall-anchored active proteinase.     

Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactis

Polymerase chain reaction (PCR) primers designed from a multiple alignment of predicted amino acid sequences from bacterial aroA genes were used to amplify a fragment of Lactococcus lactis DNA. An 8 kb fragment was then cloned from a lambda library and the DNA sequence of a 4.4 kb region determined. This region was found to contain the genes tyrA, aroA, aroK, and pheA, which are involved in aromatic amino acid biosynthesis and folate metabolism. TyrA has been shown to be secreted and AroK also has a signal sequence, suggesting that these proteins have a secondary function, possibly in the transport of amino acids. The aroA gene from L. lactis has been shown to complement an E. coli mutant strain deficient in this gene. The arrangement of genes involved in aromatic amino acid biosynthesis in L. lactis appears to differ from that in other organisms.     

Autolysis of Lactococcus lactis ssp. lactis and Lactobacillus casei ssp. casei. Cell lysis induced by a crude bacteriocin

Clinical studies indicate that patients with acute schizophrenia may benefit from benzodiazepine treatment. Therefore we investigated the benzodiazepine receptor distribution and diazepam binding in 20 patients with DSM-III schizophrenia using single photon emission computed tomography (SPECT) with iomazenil as the ligand. In each patient, two SPECT images were obtained: SPECT 1 was obtained 2 h after intravenous injection of 200 MBq I-123-iomazenil. Following SPECT 1, patients received 10 mg diazepam intravenously. Twenty min later, SPECT 2 was started. The highest iomazenil uptake was found in the occipital cortex followed by the frontal and temporal cortices. Baseline iomazenil uptake in the medial frontal cortex was significantly (P < 0.05) correlated with the BPRS total score (r = 0.46). Diazepam injection led to a significant activity decrease in iomazenil binding which was greatest in the frontal regions of interest. With respect to the medial frontal cortex, this effect was significantly (P < 0.05) more pronounced in patients with a remitting than a chronic course of the disorder. These findings suggest that changes of the benzodiazepine receptor system in the frontal cortex may be associated with severity and chronicity of schizophrenia.     

Influence of carbohydrate starvation and arginine on culturability and amino acid utilization of lactococcus lactis subsp. lactis

Two strains of Lactococcus lactis subsp. lactis were used to determine the influence of lactose and arginine on viability and amino acid use during carbohydrate starvation. Lactose provided energy for logarithmic-phase growth, and amino acids such as arginine provided energy after carbohydrate exhaustion. Survival time, cell numbers, and ATP concentrations increased with the addition of arginine to the basal medium. By the onset of lactose exhaustion, the concentrations of glycine-valine and glutamate had decreased by as much as 67% in L. lactis ML3, whereas the serine concentration increased by 97% during the same period. When no lactose was added, the concentrations of these amino acids remained constant. Similar trends were observed for L. lactis 11454. Without lactose or arginine, L. lactis ML3 was nonculturable on agar but was viable after 2 days, as measured by fluorescent viability stains and intracellular ATP levels. However, L. lactis 11454 without lactose or arginine remained culturable for at least 14 days. These data suggest that lactococci become viable but nonculturable in response to carbohydrate depletion. Additionally, these data indicate that amino acids other than arginine facilitate the survival of L. lactis during carbohydrate starvation.     

A conserved sequence in tRNA and rRNA promoters of Lactococcus lactis

A tRNA operon (trnA) from Lactococcus lactis consisting of seven tRNA genes and a 5S rRNA gene was cloned and sequenced. Promoter-fusion of the trnA promoter to a promoter-less beta-galactosidase gene of Leuconostoc mesenteroides resulted in high levels of beta-galactosidase activity in L. lactis. Searching for sequences with similarity to the sequence of the promoter region revealed a consensus sequence of promoters preceeding rRNA operons and tRNA operons from Lactococcus species including a not previously described conserved sequence (AGTT).     

Genetic manipulation of the pathway for diacetyl metabolism in Lactococcus lactis

Diacetyl is an important food flavor compound produced by certain strains of citrate-metabolizing lactic acid bacteria. Citrate is converted to pyruvate, from which diacetyl is produced via intermediate alpha-acetolactate. This paper reports the cloning and analysis of the gene (aldB) encoding alpha-acetolactate decarboxylase from Lactococcus lactis MG1363. Deletion of the MG1363 chromosomal aldB gene was achieved by double crossover homologous recombination. The mutant strain was found to produce diacetyl; alpha-acetolactate decarboxylase activity was eliminated. Overexpression of the cloned ilvBN genes (encoding an alpha-acetolactate synthase) in the aldB deletion strain produced even higher levels of alpha-acetolactate, acetoin, and diacetyl.