A functional glycogen biosynthesis pathway in Lactobacillus acidophilus: expression and analysis of the glg operon

Glycogen metabolism contributes to energy storage and various physiological functions in some prokaryotes, including colonization persistence. A role for glycogen metabolism is proposed on the survival and fitness of Lactobacillus acidophilus, a probiotic microbe, in the human gastrointestinal environment. L. acidophilus NCFM possesses a glycogen metabolism (glg) operon consisting of glgBCDAP‐amy‐pgm genes. Expression of the glg operon and glycogen accumulation were carbon source‐ and growth phase‐dependent, and were repressed by glucose. The highest intracellular glycogen content was observed in early log‐phase cells grown on trehalose, which was followed by a drastic decrease of glycogen content prior to entering stationary phase. In raffinose‐grown cells, however, glycogen accumulation gradually declined following early log phase and was maintained at stable levels throughout stationary phase. Raffinose also induced an overall higher temporal glg expression throughout growth compared with trehalose. Isogenic ΔglgA (glycogen synthase) and ΔglgB (glycogen‐branching enzyme) mutants are glycogen‐deficient and exhibited growth defects on raffinose. The latter observation suggests a reciprocal relationship between glycogen synthesis and raffinose metabolism. Deletion of glgB or glgP (glycogen phosphorylase) resulted in defective growth and increased bile sensitivity. The data indicate that glycogen metabolism is involved in growth maintenance, bile tolerance and complex carbohydrate utilization in L. acidophilus.     

Epidemic Clone I-Specific Genetic Markers in Strains of Listeria monocytogenes Serotype 4b from Foods

Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by L. monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L. monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L. monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis.     

Nuclear gene trees and the phylogenetic relationships of the mangabeys (Primates: Papionini)

Phylogenetic relationships of mangabeys within the Old World monkey tribe Papionini are inferred from analyses of nuclear DNA sequences from five unlinked loci. The following conclusions are strongly supported, based on congruence among trees derived for the five separate gene regions: (1) mangabeys are polyphyletic within the Papionini; (2) Cercocebus is the sister taxon to the genus Mandrillus; and (3) Lophocebus belongs to a clade with Papio and Theropithecus, with Papio as its most likely sister taxon. Morphologically based phylogenies positing mangabey monophyly were evaluated by mapping the sequences for each locus on these trees. The data seem to fit these trees poorly in both maximum-parsimony and likelihood analyses. Incongruence among nuclear gene trees occurred in the interrelationships among Lophocebus, Papio, and Theropithecus. Several factors that may account for this incongruence are discussed, including sampling error, random lineage sorting, and introgression.      

PPCMatrix: a PowerPC dotmatrix program to compare large genomic sequences against protein sequences

Summary : An interactive dotmatrix program for the MacOS was designed that allows comparison of DNA to protein sequences using nested 3-frame translations. Availability : Shareware, available at http://copan.bioz.unibas.ch/software/ Contact : burglin@ubaclu. unibas.ch      

The anabolic action of intermittent parathyroid hormone on cortical bone depends partly on its ability to induce nitric oxide‐mediated vasorelaxation in BALB/c mice

There is strong evidence that vasodilatory nitric oxide (NO) donors have anabolic effects on bone in humans. Parathyroid hormone (PTH), the only osteoanabolic drug currently approved, is also a vasodilator. We investigated whether the NO synthase inhibitor L‐NAME might alter the effect of PTH on bone by blocking its vasodilatory effect. BALB/c mice received 28 daily injections of PTH[1–34] (80 µg/kg/day) or L‐NAME (30 mg/kg/day), alone or in combination. Hindlimb blood perfusion was measured by laser Doppler imaging. Bone architecture, turnover and mechanical properties in the femur were analysed respectively by micro‐CT, histomorphometry and three‐point bending. PTH increased hindlimb blood flow by >30% within 10 min of injection (P < 0.001). Co‐treatment with L‐NAME blocked the action of PTH on blood flow, whereas L‐NAME alone had no effect. PTH treatment increased femoral cortical bone volume and formation rate by 20% and 110%, respectively (P < 0.001). PTH had no effect on trabecular bone volume in the femoral metaphysis although trabecular thickness and number were increased and decreased by 25%, respectively. Co‐treatment with L‐NAME restricted the PTH‐stimulated increase in cortical bone formation but had no clear‐cut effects in trabecular bone. Co‐treatment with L‐NAME did not affect the mechanical strength in femurs induced by iPTH. These results suggest that NO‐mediated vasorelaxation plays partly a role in the anabolic action of PTH on cortical bone. © 2016 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd.     

Group-specific comparison of four lactobacilli isolated from human sources using differential blast analysis

Lactic acid bacteria (LAB) have been used in fermentation processes for centuries. More recent applications including the use of LAB as probiotics have significantly increased industrial interest. Here we present a comparative genomic analysis of four completely sequenced Lactobacillus strains, isolated from the human gastrointestinal tract, versus 25 lactic acid bacterial genomes present in the public database at the time of analysis. Lactobacillus acidophilus NCFM, Lactobacillus johnsonii NCC533, Lactobacillus gasseri ATCC33323, and Lactobacillus plantarum WCFS1are all considered probiotic and widely used in industrial applications. Using Differential Blast Analysis (DBA), each genome was compared to the respective remaining three other Lactobacillus and 25 other LAB genomes. DBA highlighted strain-specific genes that were not represented in any other LAB used in this analysis and also identified group-specific genes shared within lactobacilli. Initial comparative analyses highlighted a significant number of genes involved in cell adhesion, stress responses, DNA repair and modification, and metabolic capabilities. Furthermore, the range of the recently identified potential autonomous units (PAUs) was broadened significantly, indicating the possibility of distinct families within this genetic element. Based on in silico results obtained for the model organism L. acidophilus NCFM, DBA proved to be a valuable tool to identify new key genetic regions for functional genomics and also suggested re-classification of previously annotated genes.     

Modification of Lactobacillus beta-glucuronidase activity by random mutagenesis

The Lactobacillus gasseri ADH beta-glucuronidase gene, gusA, was cloned previously and found to exhibit excellent activity in acidic pH ranges, with maximal activity at pH 5.0. In contrast, activity was limited in neutral pH ranges of 6-7. In an effort to improve the activity of the reporter enzyme in neutral pH ranges, the gusA gene was cloned into the broad host range vector, pGK12, and subjected to random mutagenesis by passage through Epicurian coli mutator strain XL1-Red. Two mutant alleles, gusA2 and gusA3, were recovered that produced beta-glucuronidase with increased activity in neutral pH ranges. One of these, gusA3, was significantly more active in the pH range of 4-8 in both Escherichia coli and L. gasseri. Sequence analysis of gusA2 and gusA3 revealed single base pair changes that resulted in D524G and D573A substitutions, respectively. The modified GusA3 enzyme has expanded potential for use as a reporter enzyme in expression hosts that are not acidophilic, as well as lactic acid bacteria and other microorganisms that grow in acidifying environments.