AbiQ, an abortive infection mechanism from Lactococcus lactis

Lactococcus lactis W-37 is highly resistant to phage infection. The cryptic plasmids from this strain were coelectroporated, along with the shuttle vector pSA3, into the plasmid-free host L. lactis LM0230. In addition to pSA3, erythromycin- and phage-resistant isolates carried pSRQ900, an 11-kb plasmid from L. lactis W-37. This plasmid made the host bacteria highly resistant (efficiency of plaquing <10(-8)) to c2- and 936-like phages. pSRQ900 did not confer any resistance to phages of the P335 species. Adsorption, cell survival, and endonucleolytic activity assays showed that pSRQ900 encodes an abortive infection mechanism. The phage resistance mechanism is limited to a 2.2-kb EcoRV/BclI fragment. Sequence analysis of this fragment revealed a complete open reading frame (abiQ), which encodes a putative protein of 183 amino acids. A frameshift mutation within abiQ completely abolished the resistant phenotype. The predicted peptide has a high content of positively charged residues (pI = 10.5) and is, in all likelihood, a cytosolic protein. AbiQ has no homology to known or deduced proteins in the databases. DNA replication assays showed that phage c21 (c2-like) and phage p2 (936-like) can still replicate in cells harboring AbiQ. However, phage DNA accumulated in its concatenated form in the infected AbiQ+ cells, whereas the AbiQ- cells contained processed (mature) phage DNA in addition to the concatenated form. The production of the major capsid protein of phage c21 was not hindered in the cells harboring AbiQ.     

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.     

Cloning, sequence and expression of the gene encoding the malolactic enzyme from Lactococcus lactis

Many lactic acid bacteria can carry out malolactic fermentation. This secondary fermentation is mediated by the NAD- and Mn(2+)-dependent malolactic enzyme, which catalyses the decarboxylation of L-malate to L-lactate. The gene we call mleS, coding for malolactic enzyme, was isolated from Lactococcus lactis. The mleS gene consists of one open reading frame capable of coding for a protein with a calculated molecular mass of 59 kDa. The amino acid sequence of the predicted MleS gene product is homologous to the sequences of different malic enzymes. Bacterial and yeast cells expressing the malolactic gene convert L-malate to L-lactate.     

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.     

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.     

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.     

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.     

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.     

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.     

Isolation, cloning, and characterisation of a trp homologue from squid (Loligo forbesi) photoreceptor membranes

The invertebrate phototransduction system is a valuable model of the ubiquitous inositol lipid signalling system. Taking advantage of the ability to obtain relatively large amounts of retinal material from the cephalopod eye, partial protein sequence data were obtained for a 92-kDa component isolated from a detergent-insensitive cytoskeletal fraction of a squid retinal microvillar membrane preparation. Degenerate oligonucleotides, designed on the basis of these sequence data, were used to isolate a full-length cDNA, encoding the 92-kDa component, using both cDNA library screening and 5'-rapid amplification of cDNA ends (5'-RACE) techniques. Comparison of the amino acid sequence encoded by this cDNA with entries in the OWL composite protein sequence database reveals greatest sequence similarity with the products of the Drosophila trp and trpl genes. Greatest variation from the Drosophila Trp protein is seen in the carboxyl-terminal region, which is considerably truncated in the squid protein and which accounts for most of the substantial difference in molecular weight seen between these proteins. This variation may be significant as the carboxyl-terminal domain has been shown to be in the regulation of several ligand-gated channels. The carboxyl-terminal domain has been expressed and shown to interact with calmodulin in a calcium-dependent fashion, thereby supporting this hypothesis. The likely occurrence of other homologues in a variety of systems suggests that this is a novel and important family of regulated ion channels involved in calcium signalling.     

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.     

Identification and characterization of the KlCMD1 gene encoding Kluyveromyces lactis calmodulin

BACKGROUND: Bronchiolitis obliterans syndrome (BOS) is the major cause of morbidity and death after lung transplantation. Therapy has focused on augmented immunosuppression with a variety of agents. Although transient responses are often achieved, sustained remission has been unusual. The outcome of cytolytic therapy for BOS at our center has been analyzed and is reported. METHODS: Between July 1988 and July 1994, 233 patients underwent lung transplantation at Barnes-Jewish Hospital. Among 207 recipients (88.8%) who survived more than 3 months, 81 recipients (39%) had development of BOS; 48 of these patients underwent 64 courses of treatment with a cytolytic agent (antilymphocyte globulin, antithymocyte globulin, or OKT3 monoclonal antibody). The cases of BOS were retrospectively analyzed to determine the impact of cytolytic therapy. RESULTS: The 4-year survival rate was significantly greater in recipients without BOS than in those with BOS (82.8% vs 46.0%; p < .05). Various clinical factors, including diagnosis, forced expiratory volume in 1 second at onset of BOS, presence or absence of pathologically proven bronchiolitis obliterans, type of transplant operation, cytomegalovirus serologic status, and cytomegalovirus pneumonia, were examined, but no significant predictor of survival after the development of BOS was discerned. The mean decrement in forced expiratory volume in 1 second was significantly reduced by cytolytic therapy (-23.5% +/- 2.3% in the 3 months before therapy vs -9.9% +/- 3.5% in the 3 months after the therapy; p < .002). Nevertheless, the stage of BOS progressed over time in spite of therapy in most cases, and only 4 recipients (4.9%) with BOS remained in a lower BOS stage 2 years after treatment. CONCLUSIONS: Recipients with BOS had a significantly lower survival rate than recipients without BOS. No predictor of survival after the onset of BOS was identified. Although cytolytic therapy decreased the rate of decline in pulmonary function in the 3 months after treatment, the stage of BOS ultimately progressed in most patients.     

Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis

The Bacillus subtilis strain VTT E-68013 was chosen for purification and characterization of its excreted phytase. Purified enzyme had maximal phytase activity at pH 7 and 55 degrees C. Isolated enzyme required calcium for its activity and/or stability and was readily inhibited by EDTA. The enzyme proved to be highly specific since, of the substrates tested, only phytate, ADP, and ATP were hydrolyzed (100, 75, and 50% of the relative activity, respectively). The phytase gene (phyC) was cloned from the B. subtilis VTT E-68013 genomic library. The deduced amino acid sequence (383 residues) showed no homology to the sequences of other phytases nor to those of any known phosphatases. PhyC did not have the conserved RHGXRXP sequence found in the active site of known phytases, and therefore PhyC appears not to be a member of the phytase subfamily of histidine acid phosphatases but a novel enzyme having phytase activity. Due to its pH profile and optimum, it could be an interesting candidate for feed applications.     

Isolation of a cytokinin gene, ZOG1, encoding zeatin O-glucosyltransferase from Phaseolus lunatus

University students who were skilled or less-skilled readers were compared on tests of auditory information processing and immediate serial recall of auditory and visual digits. Reading skill was defined by performance on a pseudoword reading task. The good readers exhibited typical modality effects with higher recall of auditory than visual items from the last 3 serial positions. On the terminal list item, the less-skilled readers showed a modality effect comparable with that of the skilled readers, but on other list items the modality effect reversed and a visual superiority was obtained. Results were discussed in terms of C. G. Penney's (1989) separate-streams model of short-term verbal memory.     

Molecular cloning, sequencing, and heterologous expression of the vaoA gene from Penicillium simplicissimum CBS 170.90 encoding vanillyl-alcohol oxidase

The cDNA encoding vanillyl-alcohol oxidase (EC 1.1.3.7) was selected from a cDNA library constructed from mRNA isolated from Penicillium simplicissimum CBS 170.90 grown on veratryl alcohol by immunochemical screening. The vaoA-cDNA nucleotide sequence revealed an open reading frame of 1680 base pairs encoding a 560-amino acid protein with a deduced mass of 62,915 Da excluding the covalently bound FAD. The deduced primary structure shares 31% sequence identity with the 8alpha-(O-tyrosyl)-FAD containing subunit of the bacterial flavocytochrome p-cresol methyl hydroxylase. The vaoA gene was isolated from a P. simplicissimum genomic library constructed in lambdaEMBL3 using the vaoA-cDNA as a probe. Comparison of the nucleotide sequence of the vaoA gene with the cDNA nucleotide sequence demonstrated that the gene is interrupted by five short introns. Aspergillus niger NW156 prtF pyrA leuA cspA transformed with the pyrA containing plasmid and a plasmid harboring the complete vaoA gene including the promoter and terminator was able to produce vaoA mRNA and active vanillyl-alcohol oxidase when grown on veratryl alcohol and anisyl alcohol. A similar induction of the vaoA gene was found for P. simplicissimum, indicating that similar regulatory systems are involved in the induction of the vaoA gene in these fungi. Introduction of a consensus ribosome binding site, AGAAGGAG, in the vaoA-cDNA resulted in elevated expression levels of active vanillyl-alcohol oxidase from the lac promoter in Escherichia coli TG2. The catalytic and spectral properties of the purified recombinant enzyme were indistinguishable from the native enzyme.