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.     

Dual Role of the Oligopeptide Permease Opp3 during Growth of Staphylococcus aureus in Milk

Staphylococcus aureus RN6390 presents a diauxic growth in milk, due to amino acid limitation. Inactivation of the oligopeptide permease Opp3 (dedicated to the nitrogen nutrition of the strain) not only affects the growth of the strain but also results in reduced expression levels of three major extracellular proteases.Staphylococcus aureus is a ubiquitous gram-positive pathogen commonly found in the environment. Some S. aureus strains are able to produce enterotoxins that cause food poisoning (6). In France, contaminated dairy products are the main source of staphylococcal food-borne disease outbreaks (3). Thus, the development of this bacterium in milk is a major concern for the safety of dairy products. Our objective was to evaluate the growth of S. aureus in milk, with special attention focused on the role of the peptide transport systems.     

An Ecological Study of Lactococci Isolated from Raw Milk in the Camembert Cheese Registered Designation of Origin Area

The genetic diversity of lactococci isolated from raw milk in the Camembert cheese Registered Designation of Origin area was studied. Two seasonal samples (winter and summer) of raw milk were obtained from six farms in two areas (Bessin and Bocage Falaisien) of Normandy. All of the strains analyzed had a Lactococcus lactis subsp. lactis phenotype, whereas the randomly amplified polymorphic DNA (RAPD) technique genotypically identified the strains as members of L. lactis subsp. lactis or L. lactis subsp. cremoris. The genotypes were confirmed by performing standard PCR with primers corresponding to a region of the histidine biosynthesis operon. The geographic distribution of each subspecies of L. lactis was determined; 80% of the Bocage Falaisien strains were members of L. lactis subsp. lactis, and 30.5% of the Bessin strains were members of L. lactis subsp. lactis. A dendrogram was produced from a computer analysis of the RAPD profiles in order to evaluate the diversity of the lactococci below the subspecies level. The coefficient of similarity for 117 of the 139 strains identified as members of L. lactis subsp. cremoris was as high as 66%. The L. lactis subsp. lactis strains were more heterogeneous and formed 10 separate clusters (the level of similarity among the clusters was 18%). Reference strains of L. lactis subsp. lactis fell into 2 of these 10 clusters, demonstrating that lactococcal isolates are clearly different. As determined by the RAPD profiles, some L. lactis subsp. lactis strains were specific to the farms from which they originated and were recovered throughout the year (in both summer and winter). Therefore, the typicality of L. lactis subsp. lactis strains was linked to the farm of origin rather than the area. These findings emphasize the significance of designation of origin and the specificity of “Camembert de Normandie” cheese.     

Slow Axonal Transport Impairment of Cytoskeletal Proteins in Streptozociti-Induced Diabetic Neuropathy

The impairment of slow axonal transport of cytoskeletal proteins was studied in the sciatic nerves of streptozocin-diabetic rats. [35S]Methionine was unilaterally injected into the fourth lumbar ganglion and spinal cord, to label the sensory and motor axons, respectively, and then the polymerized elements of the cytoskeleton and the corresponding soluble proteins were analyzed separately. In addition, the pellet/supernatant ratio for tubulin and actin was also assessed. Our results indicate that the velocity of slow component a (SCa) of axonal transport, particularly that of neurofilaments, was strongly reduced (by 60%) in sensory axons. At the same time, a decreased pellet/supernatant ratio of tubulin, possibly owing to a depolymerization of stable microtubules, was also observed. The transport of slow component b (SCb) of axonal transport was also impaired, but the extent of this impairment could not be precisely evaluated. In contrast, motor axons showed little or no impairment of both SCa and SCb at the time studied, a result suggesting a delayed development of the neuropathy in motor axons.     

Slow Recovery from Local Disturbances as an Indicator for Loss of Ecosystem Resilience

A range of indicators have been proposed for identifying the elevated risk of critical transitions in ecosystems. Most indicators are based on the idea that critical slowing down can be inferred from changes in statistical properties of natural fluctuations and spatial patterns. However, identifying these signals in nature has remained challenging. An alternative approach is to infer changes in resilience from differences in standardized experimental perturbations. However, system-wide experimental perturbations are rarely feasible. Here we evaluate the potential to infer the risk of large-scale systemic transitions from local experimental or natural perturbations. We use models of spatially explicit landscapes to illustrate how recovery rates upon small-scale perturbations decrease as an ecosystem approaches a tipping point for a large-scale collapse. We show that the recovery trajectory depends on: (1) the resilience of the ecosystem at large scale, (2) the dispersal rate of organisms, and (3) the scale of the perturbation. In addition, we show that recovery of natural disturbances in a heterogeneous environment can potentially function as an indicator of resilience of a large-scale ecosystem. Our analyses reveal fundamental differences between large-scale weak and local-scale strong perturbations, leading to an overview of opportunities and limitations of the use of local disturbance-recovery experiments.     

Transport of 14C-IAA in Cucurbita maxima: A Comparative Study of Rapid, Non-Polar Movement and Slow, Polar Auxin Transport

The transport of ¹⁴C-IAA has been studied in Cucurbita maxima.IAA fed to the leaf of an intact plant moves rapidly in a non-polarfashion in the phloem. Collection and analysis of exudate fromsevered sieve tubes showed that there was no metabolic conversionor complexing of IAA for several hours. Polar movement of ¹⁴C-IAA in isolated internode segments occursat rates an order of magnitude slower than movement in the phloem.The importance of discrete and isolated channels of hormonetransport, that vary in direction and rates, is briefly discussed.     

Modulation of Activity of One Neuron by Subthreshold Slow Potentials in Another in Lobster Cardiac Ganglion

A direct demonstration is given of interaction between specific neurons without impulses, via graded slow potentials electrotonically spread from one cell to another. Repetitive polarizing or depolarizing current pulses of 50 to 200 msec. and subthreshold intensity were passed through an intracellular electrode in the soma of a follower cell of the isolated ganglion. When the frequency is near the natural rhythm of impulse bursts corresponding to heart beats and arising in a pacemaker cell 5 to 10 mm. posteriorly, the bursts rapidly become synchronized with the pulses. The effect disappears upon withdrawing the intracellular electrode. Brief pulses or full spikes in the follower are not effective. Hyperpolarizing long pulses attract the burst to a fixed period after the end of the pulse, depolarizations after the beginning of the pulse. The natural rhythm promptly reappears when the pulses are stopped and occasionally breaks through during weak repetitive pulses. Current pulses in postsynaptic cells also alter the threshold of a presynaptic neuron to externally applied stimuli. Some kind of direct, low resistance pathway for electrotonic spread, discriminating against spikes because of their brevity, is inferred, providing a basis for subthreshold interaction which is specific and not by way of a field effect. Due to the sensitivity of modulation of ongoing rhythms, electrotonic currents can be effective even after decrementing over several millimeters.     

Development of a Plasmid-Mediated Reporter System for In Vivo Monitoring of Gene Expression in the Archaeon Methanosarcina acetivorans

A plasmid-based gene reporter system has been developed to construct lacZ gene fusions for monitoring intrinsic promoter expression in Methanosarcina acetivorans. Constructs transform with high efficiency that can be readily screened by color selection on plates and exhibit a consistent copy number on different substrates negating the need for gene copy normalization. Expression of the CO dehydrogenase-acetyl coenzyme A synthase promoter fusion to lacZ revealed 18- to 54-fold down-regulation in cells grown on methylotrophic substrates compared with acetate-grown cells, which is up to an order of magnitude greater than the range of regulation previously reported by enzyme activity assays. This system complements and expands the current techniques for studying genetics of the methanosarcinal Archaea by providing a rapid method for monitoring and quantifying gene expression.     

Axonal Transport of Slow Component a in Sciatic Nerves of Hypo-and Hyperthyroid Rats

Axonal transport of slow component a was studied in dorsal root afferents of the sciatic nerves of hypo- and hyperthyroid rats. Three experimental groups of rats were made hypothyroid at the age of 12 weeks by the administration of 131I. From the age of 22 weeks to the end of the study, the groups were treated with daily subcutaneous injections of thyroxine in various doses to make them hypo-(0 microgram/100 g), normo- (1 microgram/100 g), and hyperthyroid (6 micrograms/100 g), respectively. The hypothyroid group had a moderate thyroid hormone deficiency (a serum triiodothyronine level of 0.19 +/- 0.10 nmol/L and a heart/body weight ratio of 1.87 +/- 0.09 g/kg at time of killing compared with 0.60 +/- 0.09 nmol/L and 2.18 +/- 0.06 g/kg, respectively, for the control group). The hyperthyroid group was severely deranged, with serum triiodothyronine being 3.30 +/- 0.37 nmol/L and a heart/body weight ratio of 3.11 +/- 0.16 g/kg. The hypothyroid rats showed a reduction in mean velocity for the transport of slow component a (0.80 +/- 0.07 mm/day compared with 0.91 +/- 0.05 mm/day in the controls). The width of the wave of activity was smaller for the hyperthyroid group than for the control group (6.6 +/- 0.7 mm compared with 8.1 +/- 1.2 mm), suggesting an increased clearance of the axonally transported activity in the proximal axon. A decrease in transport of slow component a in hypothyroidism may be the explanation of peripheral neuropathy with axonal degeneration occasionally seen in patients with severe myxoedema.     

Dipeptidyl Aminopeptidase III of Guinea-Pig Brain: Specificity for Short Oligopeptide Sequences

Abstract: A dipeptidyl aminopeptidase III-type activity has been purified from the cytoplasm of guinea-pig brain using arginyl-arginyl-7-amido-4 methylcoumarin as substrate. The enzyme was purified 754-fold relative to the crude homogenate and with a 12.7% recovery. The purified enzyme was found to have a relative molecular weight of 85,000 and consists of one polypeptide chain of relative molecular weight 80,000, on the basis of its migration on calibrated sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel. It is highly sensitive to the presence of chelating agents, sulphydryl reactive agents, and the dipeptide Tyr-Tyr. Dithiothreitol (1 m M ) reduced activity by 28%, and 36 and 65% inhibition was noted with phenylmethylsulphonyl fluoride and puromycin (both at 1 m M ), respectively. Little or no inhibition was observed with bestatin, bacitracin, captopril, amastatin, and arphamenine B. The purified enzyme released dipeptide moieties from a wide range of peptides including enkephalin sequences and also angiotensin sequences up to the octapeptide angiotensin II. These sequences inhibited the hydrolysis of arginyl-arginyl-7-amido-4-methylcoumarin by dipeptidyl aminopeptidase III with K _i values in the micromolar range. No hydrolysis was observed with angiotensin I or with peptide sequences containing more than 10 amino acids. No hydrolysis was observed also with peptide sequences containing a Pro residue on either side of the sissile bond. Peptides containing less than four amino acids were not hydrolysed.     

Slow potential changes in mammalian muscle fibers during prolonged hyperpolarization: Transport number effects and chloride depletion

Summary Mammalian skeletal muscle fibers exhibit large slow changes in membrane potential when hyperpolarized in standard chloride solutions. These large slow potential changes are radically reduced in low chloride solutions, where the faster and smaller potential change (creep), usually observed in amphibian fibers, becomes apparent. The slow potential change during a hyperpolarizing current pulse leads to an increase in apparent resistance of up to nine times the instantaneous value and takes minutes to reach a steady value. It then takes a similar time to decay very slowly back to the resting membrane potential after the current pulse. The halftime for the slow potential change was found to be inversely proportional to the current magnitude. From measurements of immediate postpulse membrane potentials, assuming constant ionic permeabilities, the internal chloride concentration was calculated to decrease exponentially towards a steady value (e.g., for one fiber from 12.3 to 6.6mm after a 330-sec pulse). The time course and magnitude of the concentration change were predicted from chloride transport number differences, and the known and measured properties of the fibers, and were found to agree very well with the values obtained from experimental measurements. In addition, the shapes of theV ₂-V ₁ responses, measured in the three-electrode current clamp set-up with either potassium chloride or potassium citrate current electrodes, were as predicted by transport number chloride depletion effects and were at variance with the predictions of a permeability change mechanism.     

Transport System for Citric Acid in Proteus vulgaris

Strains of Proteus vulgaris utilized only citric acid among the tricarboxylic acids. Citric acid uptake in P. vulgaris IFO 3045 was mediated by a single transport system induced by citrate, isocitrate or cis-aconitate and possessed a weak affinity to isocitric acid. This citric acid transport system corresponds to the system of Salmonella typhimurium or Serratia marce-scens, which is induced by citrate, isocitrate or cis-aconitate and transports citric and isocitric acids.     

Diauxic Growth of Azotobacter vinelandii on Galactose and Glucose: Regulation of Glucose Transport by Another Hexose

The growth curve of Azotobacter vinelandii was biphasic when the organism was grown in a medium containing a mixture of galactose and glucose. Galactose was the primary carbon source; glucose was also consumed, but the rate at which it was consumed was lower than the rate at which galactose was consumed during the first phase of growth. Metabolic pathways for both sugars were induced. Cell cultures exhibited a second lag period as galactose was depleted. The length of this lag phase varied from 2 to 10 h depending on the pregrowth history of the cells. The second log growth phase occurred at the expense of the remaining glucose in the medium and was accompanied by induction of the high-maximum rate of metabolism glucose-induced glucose permease and increases in the levels of glucose metabolic enzymes. The second lag phase of diauxie may have been due to the time required for induction of the glucose-induced glucose permease.     

Autolysis of Lactococci: Detection of Lytic Enzymes by Polyacrylamide Gel Electrophoresis and Characterization in Buffer Systems

Lactococcal strains were screened for bacteriolytic activity against Micrococcus luteus cells, lactococcal cells, and cell walls. Thirty strains were screened for bacteriolytic activity against cells and cell walls incorporated into agar medium. Enzymes from all strains hydrolyzed the substrates; however, the activity against Micrococcus cells was much higher than against Lactococcus cells or cell walls. Electrophoretic profiles of bacteriolytic activities of culture supernatants, sodium dodecyl sulfate-treated cell extracts, cell wall fractions, and cell extracts were analyzed in sodium dodecyl sulfate-polyacrylamide gels containing M. luteus cells or lactococcal cell walls as the substrate. The 22 strains tested contained two to five lytic bands in the culture supernatant, ranging in size between 32 and 53 kDa. The cell extracts, the sodium dodecyl sulfate-treated cell extracts, and the cell wall fractions revealed two lytic bands of 47 and 53 kDa. Effects of external factors on autolysis of some strains were determined in buffer systems. Optimal autolysis was observed in the exponential growth phase at pH 6.0 to 7.5 and at a temperature of 30(deg)C. Two of three strains tested seemed to contain a glycosidase, and all three strains contained an N-acetylmuramyl-l-alanine amidase or an endopeptidase.     

Plasmid-Mediated AmpC: Prevalence in Community-Acquired Isolates in Amsterdam,the Netherlands,and Risk Factors for Carriage

Objectives

The objective of this study was to determine the prevalence of pAmpC beta-lactamases in community-acquired Gram negative bacteria in the Netherlands, and to identify possible risk factors for carriage of these strains.

Methods

Fecal samples were obtained from community-dwelling volunteers. Participants also returned a questionnaire for analysis of risk factors. Screening for pAmpC was performed with selective enrichment broth and a selective screening agar. Confirmation of AmpC-production was performed with two double disc combination tests: cefotaxime and ceftazidime with either boronic acid or cloxacillin as inhibitor. Multiplex PCR was used as gold standard for detection of pAmpC. 16S rRNA PCR and AFLP were performed as required, plasmids were identified by PCR-based replicon typing. Questionnaire results were analyzed with SPSS, version 20.0.

Results

Fecal samples were obtained from 550 volunteers; mean age 51 years (range: 18–91), 61% were females. pAmpC was present in seven E. coli isolates (7/550, 1.3%, 0.6–2.7 95% CI): six CMY-2-like pAmpC and one DHA. ESBL-encoding genes were found in 52/550 (9.5%, 7.3–12.2 95% CI) isolates; these were predominantly blaCTX-M genes. Two isolates had both ESBL and pAmpC. Admission to a hospital in the previous year was the only risk factor we identified.

Conclusions

Our data indicate that the prevalence of pAmpC in the community seems still low. However, since pAmpC-producing isolates were not identified as ESBL producers by routine algorithms, there is consistent risk that further increase of their prevalence might go undetected.