Comparison of Campylobacter jejuni isolates from human, food, veterinary and environmental sources in Iceland using PFGE, MLST and fla-SVR sequencing

Aims: To evaluate the probiotic properties of strains isolated from smoked salmon and previously identified as bacteriocin producers. Methods and Results: Strains Lactobacillus curvatus ET06, ET30 and ET31, Lactobacillus fermentum ET35, Lactobacillus delbrueckii ET32, Pediococcus acidilactici ET34 and Enterococcus faecium ET05, ET12 and ET88 survived conditions simulating the gastrointestinal tract (GIT) and produced bacteriocins active against several strains of Listeria monocytogenes, but presented very low activity against other lactic acid bacteria (LAB). Cell‐free supernatants containing bacteriocins, added to 3‐h‐old cultures of L. monocytogenes 603, suppressed growth over 12 h. Auto‐aggregation was strain‐specific, and values ranged from 7·2% for ET35 to 12·1% for ET05. Various degrees of co‐aggregation with L. monocytogenes 603, Lactobacillus sakei ATCC 15521 and Enterococcus faecalis ATCC 19443 were observed. Adherence of the bacteriocinogenic strains to Caco‐2 cells was within the range reported for Lactobacillus rhamnosus GG, a well‐known probiotic. The highest levels of hydrophobicity were recorded for Lact. curvatus (61·9–64·6%), Lact. fermentum (78·9%), Lact. delbrueckii (43·7%) and Ped. acidilactici (51·3%), which are higher than the one recorded for Lact. rhamnosus GG (53·3%). These strains were highly sensitive to several antibiotics and affected by several drugs from different generic groups in a strain‐dependent manner. Conclusions: Smoked salmon is a rich source of probiotic LAB. All strains survived conditions simulating the GIT and produced bacteriocins active against various pathogens. Adherence to Caco‐2 cells was within the range reported for Lact. rhamnosus GG, a well‐known probiotic. In addition, the high hydrophobicity readings recorded define the strains as good probiotics. Significance and Impact of the Study: Smoked salmon contains a number of different probiotic LAB and could be marketed as having a potential beneficial effect.     

Probiotic potential of lactic acid bacteria isolated from chicken gastrointestinal digestive tract

This study was conducted in order to evaluate the probiotic properties of lactic acid bacteria (LAB) isolated from intestinal tract of broilers and Thai indigenous chickens. The major properties, including the gastric juice and bile salts tolerance, starch, protein and lipid digesting capabilities, and the inhibition on certain pathogenic bacteria were investigated. Three-hundred and twenty-two and 226 LAB strains were isolated from ten broilers and eight Thai indigenous chickens, respectively. The gastrointestinal transit tolerance of these 548 isolates was determined by exposing washed cell suspension at 41°C to simulated gastric juice (pH 2.5) containing pepsin (3 mg ml⁻¹), and to simulated small intestinal juice (pH 8.0) in the presence of pancreatin (1 mg ml⁻¹) and 7% fresh chicken bile, mimicking the gastrointestinal environment. The survival of 20 isolates was found after passing through the gastrointestinal conditions. The survival rates of six strains; KT3L20, KT2CR5, KT10L22, KT5S19, KT4S13 and PM1L12 from the sequential study were 43.68, 37.56, 33.84, 32.89, 31.37 and 27.19%, respectively. Twelve isolates exhibited protein digestion on agar plate but no isolates showed the ability to digest starch and lipid. All 20 LAB showed the antimicrobial activity against Salmonella sp., Staphylococcus aureus and Escherichia coli except one strain which did not show the inhibitory activity toward E. coli. Accordingly, five isolates of selected LAB (KT2L24, KT3L20, KT4S13, KT3CE27 and KT8S16) can be classified as the best probiotics and were identified as Enterococcus faecalis, Enterococcus durans, Enterococcus faecium, Pediococcus pentosaceus, and Enterococcus faecium, respectively. The survival rate of microencapsulation of E. durans KT3L20 under simulated small intestine juice after sequential of simulated gastric juice was also investigated. An extrusion technique exhibited a higher survival rate than emulsion technique and free cell, respectively.     

Isolation and basic characterization of a β‐glucosidase from a strain of Lactobacillus brevis isolated from a malolactic starter culture

Aims: To study glycosidase activities of a Lactobacillus brevis strain and to isolate an intracellular β‐glucosidase from this strain. Methods and Results: Lactic acid bacteria (LAB) isolated from a commercially available starter culture preparation for malolactic fermentation were tested for β‐glycosidase activities. A strain of Lact. brevis showing high intracellular β‐d ‐glucosidase, β‐d ‐xylosidase and α‐l ‐arabinosidase activities was selected for purification and characterization of its β‐glucosidase. The pure glucosidase from Lact. brevis has also side activities of xylosidase, arabinosidase and cellobiosidase. It is a homotetramer of 330 kDa and has an isoelectric point at pH 3·5. The K_m for p‐nitrophenyl‐β‐d ‐glucopyranoside and p‐nitrophenyl‐β‐d ‐xylopyranoside is 0·22 and 1·14 mmol l^?1, respectively. The β‐glucosidase activity was strongly inhibited by gluconic acid δ‐lactone, partially by glucose and gluconate, but not by fructose. Ethanol and methanol were found to increase the activity up to twofold. The free enzyme was stable at pH 7·0 (t_1/2 = 50 day) but not at pH 4·0 (t_1/2 = 4 days). Conclusions: The β‐glucosidase from Lact. brevis is widely different to that characterized from Lactobacillus casei ( Coulon et al. 1998 ) and Lactobacillus plantarum ( Sestelo et al. 2004 ). The high tolerance to fructose and ethanol, the low inhibitory effect of glucose on the enzyme activity and the good long‐term stability could be of great interest for the release of aroma compounds during winemaking. Significance and Impact of the study: Although the release of aroma compounds by LAB has been demonstrated by several authors, little information exists on the responsible enzymes. This study contains the first characterization of an intracellular β‐glucosidase isolated from a wine‐related strain of Lact. brevis.     

Crystallographic and mutational analyses of cystathionine β‐synthase in the H2S‐synthetic gene cluster in Lactobacillus plantarum

Cystathionine β‐synthase (CBS) catalyzes the formation of l ‐cystathionine from l ‐serine and l ‐homocysteine. The resulting l ‐cystathionine is decomposed into l ‐cysteine, ammonia, and α‐ketobutylic acid by cystathionine γ‐lyase (CGL). This reverse transsulfuration pathway, which is catalyzed by both enzymes, mainly occurs in eukaryotic cells. The eukaryotic CBS and CGL have recently been recognized as major physiological enzymes for the generation of hydrogen sulfide (H₂S). In some bacteria, including the plant‐derived lactic acid bacterium Lactobacillus plantarum, the CBS‐ and CGL‐encoding genes form a cluster in their genomes. Inactivation of these enzymes has been reported to suppress H₂S production in bacteria; interestingly, it has been shown that H₂S suppression increases their susceptibility to various antibiotics. In the present study, we characterized the enzymatic properties of the L. plantarum CBS, whose amino acid sequence displays a similarity with those of O‐acetyl‐l ‐serine sulfhydrylase (OASS) that catalyzes the generation of l ‐cysteine from O‐acetyl‐l ‐serine (l ‐OAS) and H₂S. The L. plantarum CBS shows l ‐OAS‐ and l ‐cysteine‐dependent CBS activities together with OASS activity. Especially, it catalyzes the formation of H₂S in the presence of l ‐cysteine and l ‐homocysteine, together with the formation of l ‐cystathionine. The high affinity toward l ‐cysteine as a first substrate and tendency to use l ‐homocysteine as a second substrate might be associated with its enzymatic ability to generate H₂S. Crystallographic and mutational analyses of CBS indicate that the Ala70 and Glu223 residues at the substrate binding pocket are important for the H₂S‐generating activity.     

Inhibitory effect of different chicken-derived lactic acid bacteria isolates on drug resistant Salmonella SE47 isolated from eggs

Lactic Acid Bacteria (LAB) regulate and maintain the stability of healthy microbial flora, inhibit the adhesion of pathogenic bacteria and promote the colonization of beneficial micro-organisms. The drug resistance and pathogenicity of Salmonella enteritis SE47 isolated from retail eggs were investigated. Meanwhile, Enterococcus faecalis L76 and Lactobacillus salivarius LAB35 were isolated from intestine of chicken. With SE47 as indicator bacteria, the diameters of L76 and LAB35 inhibition zones were 12 mm and 8·5 mm, respectively, by agar inhibition circle method, which indicated that both of them had inhibitory effect on Salmonella, and L76 had better antibacterial effect; two chicken-derived lactic acid bacteria isolates and Salmonella SE47 were incubated with Caco-2. The adhesion index of L76 was 17·5%, which was much higher than that of LAB35 (10·21%) and SE47 (4·89%), this experiment shows that the higher the bacteriostatic effect of potential probiotics, the stronger the adhesion ability; then Caco-2 cells were incubated with different bacteria, and the survival of Caco-2 cells was observed by flow cytometry. Compared with Salmonella SE47, the results showed that lactic acid bacteria isolates could effectively protect Caco-2 cells; finally, after different bacteria incubated Caco-2 cells, according to the cytokine detection kit, the RNA of Caco-2 cells was extracted and transcribed into cDNA, then detected by fluorescence quantitative PCR, the results showed that L76 could protect Caco-2 cells from the invasion of Salmonella SE47, with less cell membrane rupture and lower expression of MIF and TNF genes. Therefore, the lactic acid bacteria isolates can effectively inhibit the adhesion of Salmonella and protect the integrity of intestinal barrier.     

Controlled feeding of hydrogen peroxide as oxygen source improves production of 5-ketofructose From L-sorbose using engineered pyranose 2-oxidase from Peniophora gigantea

5‐Keto‐D ‐fructose is a useful starting material for the synthesis of pyrrolidine iminosugars. It can be prepared by regioselective oxidation of L ‐sorbose using pyranose 2‐oxidase (P2Ox) and O₂ as a cosubstrate. As the solubility of O₂ in aqueous solution is low and the affinity of P2Ox for O₂ is poor, we developed a new and efficient process for the production of 5‐keto‐D ‐fructose based on engineered P2Ox from Peniophora gigantea and in situ generation of O₂ from H₂O₂ with catalase. This kind of oxygen supply required efficient mixing of the bioreactor which was achieved by controlled feeding of H₂O₂ close to the impeller tip where energy dissipation rate is highest. Thus bubbling, known to affect enzyme stability, was largely avoided, and the process could be run up to 145% oxygen super‐saturation which speeds‐up P2Ox activity. Under these conditions quantitative oxidation of 180 g L^?1 L ‐sorbose to 5‐keto‐D ‐fructose could be achieved within 4 h, resulting in a threefold higher overall productivity of the process compared to a process using gaseous oxygen supply. In addition, in situ generation of O₂ from H₂O₂ lowered the oxygen demand of the process by a factor of 100 compared to gaseous oxygen supply. Biotechnol. Bioeng. 2012; 109: 2941–2945. © 2012 Wiley Periodicals, Inc.     

Functional roles of superoxide and hydrogen peroxide generated by mitochondrial DNA mutation in regulating tumorigenicity of HepG2 cells

Mitochondria are a major source of reactive oxygen species (ROS). Recent studies have estimated that mitochondrial DNA mutations inducing the overproduction of ROS are associated with human cancer. However, a substantial challenge in elucidating their diverse roles in regulating tumorigenesis is the lack of methods for probing ROS in living systems with molecular specificity. In this study, we reported the application of two fluorescent probes, 2‐chloro‐1,3‐dibenzothiazolinecyclohexene and naphthofluorescein disulfonate, which showed high selectivity for superoxide (O₂^•−) and hydrogen peroxide (H₂O₂). They were capable of detecting and visualizing O₂^•− and H₂O₂ overproduction caused by a mutation in the gene encoding nicotinamide adenine dinucleotide dehydrogenase subunit 6 (ND6) in HepG2 cells. The levels of O₂^•− and H₂O₂ in mitochondria isolated from HepG2 cells were found to be 0·63 ± 0·07 and 1·13 ± 0·05 μM, respectively. Using assays of tumorigenesis in mouse models, we found that treatment of the mice with different ROS scavengers suppressed tumour growth. These findings suggested that ROS generated by ND6 gene mutation do play an important role in regulating tumorigenesis and H₂O₂ may be a key modulator. Copyright © 2011 John Wiley & Sons, Ltd.     

Removal of patulin from apple juice using inactivated lactic acid bacteria

Aims: Apples and apple products are the most notably commodities contaminated with patulin (PAT), which cause detrimental effects on human health and economic problems. The primary objective of this study was to investigate the removal of PAT contamination from apple juice using 10 different inactivated lactic acid bacteria (LAB) strains. Methods and Results: Significant quantities of PAT ranging from 47 to 80% were bound to all tested bacterial strains, whereas Lactobacillus rhamnosus 6224 and Enterococcus faecium 21605 caused a decrease of PAT by 80·4 and 64·5%, respectively. The results showed that the binding of PAT depends on the initial concentration of toxin and the adsorption temperature, also the differences in biomass existed among the 10 bacterial strains. IR analysis was performed to identify potential functional groups and the possible binding sites related to PAT adsorption. Conclusions: The removal of PAT was observed to be strain specific. The results indicated that the biosorption process did not affect the quality of juice. FTIR analysis showed that the cell wall plays a key role in PAT adsorption. Significance and Impact of the Study: Our results proof that inactivated LAB have the potential as a novel and promising adsorbent to bind PAT effectively.     

l‐cysteine desulfhydrase‐related H2S production is involved in OsSE5‐promoted ammonium tolerance in roots of Oryza sativa

Previous studies revealed that rice heme oxygenase PHOTOPERIOD SENSITIVITY 5 (OsSE5) is involved in the regulation of tolerance to excess ammonium by enhancing antioxidant defence. In this study, the relationship between OsSE5 and hydrogen sulfide (H₂S), a well‐known signalling molecule, was investigated. Results showed that NH₄Cl triggered the induction of l ‐cysteine desulfhydrase (l ‐DES)‐related H₂S production in rice seedling roots. A H₂S donor not only alleviated the excess ammonium‐triggered inhibition of root growth but also reduced endogenous ammonium, both of which were aggravated by hypotaurine (HT, a H₂S scavenger) or dl ‐propargylglycine (PAG, a l ‐DES inhibitor). Nitrogen metabolism‐related enzymes were activated by H₂S, thus resulting in the induction of amino acid synthesis and total nitrogen content. Interestingly, the activity of l ‐DES, as well as the enzymes involved in nitrogen metabolism, was significantly increased in the OsSE5‐overexpression line (35S:OsSE5), whereas it impaired in the OsSE5‐knockdown mutant (OsSE5‐RNAi). The application of the HT/PAG or H₂S donor could differentially block or rescue NH₄Cl‐hyposensitivity or hypersensitivity phenotypes in 35S:OsSE5‐1 or OsSE5‐RNAi‐1 plants, with a concomitant modulation of nitrogen assimilation. Taken together, these results illustrated that H₂S function as an indispensable positive regulator participated in OsSE5‐promoted ammonium tolerance, in which nitrogen metabolism was facilitated.     

Polyphasic taxonomic studies of lactic acid bacteria associated with Tunisian fermented meat based on the heterogeneity of the 16S–23S rRNA gene intergenic spacer region

The objective of this work was to investigate the structure and diversity of lactic acid bacteria (LAB) communities in traditionally fermented meat collected from different areas of Tunisia. A polyphasic study, which involves phenotypic tests and ribosomal DNA-based techniques, was used to identify Gram-positive and catalase-negative isolates. PCR amplification of the 16S–23S rDNA ISR of 102 isolates and other reference LAB strains gave (1) one type of rrn operon (M-ISR) for lactococci, (2) two types of rrn operon (S-ISR and M-ISR) for enterococci, (3) two types of rrn operon (S-ISR and L-ISR) for Lactobacilli, and (4) three PCR amplicons (S-ISR, M-ISR, and L-ISR) obtained for Pediococcus spp. and Weissella genus. The clustering and comparison of ISR–RFLP profiles given by the isolates with those given by reference LAB strains, allowed their identification as Lactococcus lactis, Enterococcus faecium, Enterococcus faecalis, Enterococcus sanguinicola, Enterococcus hawaiiensis, Lactobacillus sakei, Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus alimentarius, Pediococcus pentosaceus, and Weissella confusa. Combined 16S–23S rDNA ISR and RFLP patterns can be considered as a good potential target for a rapid and reliable differentiation between isolates of LAB and provided further information on the organization of their rrn operons.     

Role of the cystathionine γ lyase/hydrogen sulfide pathway in human melanoma progression

In humans, two main metabolic enzymes synthesize hydrogen sulfide (H₂S): cystathionine γ lyase (CSE) and cystathionine β synthase (CBS). A third enzyme, 3‐mercaptopyruvate sulfurtransferase (3‐MST), synthesizes H₂S in the presence of the substrate 3‐mercaptopyruvate (3‐MP). The immunohistochemistry analysis performed on human melanoma samples demonstrated that CSE expression was highest in primary tumors, decreased in the metastatic lesions and was almost silent in non‐lymph node metastases. The primary role played by CSE was confirmed by the finding that the overexpression of CSE induced spontaneous apoptosis of human melanoma cells. The same effect was achieved using different H₂S donors, the most active of which was diallyl trisulfide (DATS). The main pro‐apoptotic mechanisms involved were suppression of nuclear factor‐κB activity and inhibition of AKT and extracellular signal‐regulated kinase pathways. A proof of concept was obtained in vivo using a murine melanoma model. In fact, either l ‐cysteine, the CSE substrate, or DATS inhibited tumor growth in mice. In conclusion, we have determined that the l ‐cysteine/CSE/H₂S pathway is involved in melanoma progression.     

Novel vitamin B<Subscript>12</Subscript>-producing <Emphasis Type="Italic">Enterococcus</Emphasis> spp. and preliminary in vitro evaluation of probiotic potentials

Vitamin B₁₂ is an essential nutrient required for crucial metabolic processes in humans. Vitamin B₁₂-producing lactic acid bacteria (LAB) have been attracting increased attentions currently because of the generally recognized as safe (GRAS) status. Most of recent studies focused on Lactobacillus, and little is known about B₁₂-producing Enterococcus. In the present study, five Enterococcus strains isolated from infant feces were identified as vitamin B₁₂ producers. Among them, Enterococcus faecium LZ86 had the highest B₁₂ production (499.8 ± 83.7 μg/L), and the B₁₂ compound from LZ86 was identified as the biological active adenosylcobalamin, using reversed phase high-performance liquid (RP-HPLC) chromatogram. We examined basic probiotic and safety properties of E. faecium LZ86 and found that it was able to survive harsh environmental conditions (hot temperature, cold temperature, ethanol and osmotic stresses), tolerate gastric acid (pH 2.0, 3 h) and bile salts (0.3%), and adhere to Caco-2 cells. We also showed that E. faecium LZ86 is devoid of transferable antibiotic resistance and potential virulence factors. Together, here we report a B₁₂-producing E. faecium strain LZ86 firstly, which has desirable probiotic properties and may serve as a good candidate for vitamin B₁₂ fortification in food industry.     

Structural insights into catalysis by βC-S lyase from Streptococcus anginosus

Hydrogen sulfide (H₂S) is a causative agent of oral malodor and may play an important role in the pathogenicity of oral bacteria such as Streptococcus anginosus. In this microorganism, H₂S production is associated with βC‐S lyase (Lcd) encoded by lcd gene, which is a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme that catalyzes the α,β‐elimination of sulfur‐containing amino acids. When Lcd acts on L ‐cysteine, H₂S is produced along with pyruvate and ammonia. To understand the H₂S‐producing mechanism of Lcd in detail, we determined the crystal structures of substrate‐free Lcd (internal aldimine form) and two reaction intermediate complexes (external aldimine and α‐aminoacrylate forms). The formation of intermediates induced little changes in the overall structure of the enzyme and in the active site residues, with the exception of Lys234, a PLP‐binding residue. Structural and mutational analyses highlighted the importance of the active site residues Tyr60, Tyr119, and Arg365. In particular, Tyr119 forms a hydrogen bond with the side chain oxygen atom of L ‐serine, a substrate analog, in the external aldimine form suggesting its role in the recognition of the sulfur atom of the true substrate (L ‐cysteine). Tyr119 also plays a role in fixing the PLP cofactor at the proper position during catalysis through binding with its side chain. Finally, we partly modified the catalytic mechanism known for cystalysin, a βC‐S lyase from Treponema denticola, and proposed an improved mechanism, which seems to be common to the βC‐S lyases from oral bacteria. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Specific enumeration of lactic acid bacteria in ragi tape by colony hybridization with specific oligonucleotide probes

Lactic acid bacteria (LAB) are among the integral microflora of ragi tape, a dry starter of Balinese rice wine, brem. The species diversity and population level of LAB present in different types of ragi tape were studied by colony hybridization using 16S and 23S rDNA targeted oligonucleotide probes. These probes were DB6, Lbc, Wgp, and Rpt, which were specific for Enterococcus faecium, Lactobacillus curvatus, Weissella spp., and Pediococcus pentosaceus, respectively. Results revealed that P. pentosaceus and Weissella spp. were the predominant LAB in ragi tape, whereas L. curvatus and E. faecium were associated specific to types of ragi tape. A 21-mer species-specific oligonucleotide probe, Rpt, that targets the 16S rDNA of P. pentosaceus was developed in this study and found to be highly specific to be used as an effective tool to enumerate population of this species in ragi tape and its population changes during rice wine production. It was detected that LAB showed active growth during the early stage of brem fermentation. A succession of growth of LAB population during the fermentation was observed in which the heterofermentative LAB, Weissella spp., grew first, followed by the proliferation of P. pentosaceus.     

The microbiology of Bandji,palm wine of Borassus akeassii from Burkina Faso: identification and genotypic diversity of yeasts,lactic acid and acetic acid bacteria

Aim

To investigate physicochemical characteristics and especially genotypic diversity of the main culturable micro‐organisms involved in fermentation of sap from Borassus akeassii, a newly identified palm tree from West Africa.

Methods and Results

Physicochemical characterization was performed using conventional methods. Identification of micro‐organisms included phenotyping and sequencing of: 26S rRNA gene for yeasts, 16S rRNA and gyrB genes for lactic acid bacteria (LAB) and acetic acid bacteria (AAB). Interspecies and intraspecies genotypic diversities of the micro‐organisms were screened respectively by amplification of the ITS1‐5.8S rDNA‐ITS2/16S‐23S rDNA ITS regions and repetitive sequence‐based PCR (rep‐PCR). The physicochemical characteristics of samples were: pH: 3·48–4·12, titratable acidity: 1·67–3·50 mg KOH g^?1, acetic acid: 0·16–0·37%, alcohol content: 0·30–2·73%, sugars (degrees Brix): 2·70–8·50. Yeast included mainly Saccharomyces cerevisiae and species of the genera Arthroascus, Issatchenkia, Candida, Trichosporon, Hanseniaspora, Kodamaea, Schizosaccharomyces, Trigonopsis and Galactomyces. Lactobacillus plantarum was the predominant LAB species. Three other species of Lactobacillus were also identified as well as isolates of Leuconostoc mesenteroides, Fructobacillus durionis and Streptococcus mitis. Acetic acid bacteria included nine species of the genus Acetobacter with Acetobacter indonesiensis as predominant species. In addition, isolates of Gluconobacter oxydans and Gluconacetobacter saccharivorans were also identified. Intraspecies diversity was observed for some species of micro‐organisms including four genotypes for Acet. indonesiensis, three for Candida tropicalis and Lactobacillus fermentum and two each for S. cerevisiae, Trichosporon asahii, Candida pararugosa and Acetobacter tropicalis.

Conclusion

fermentation of palm sap from B. akeassii involved multi‐yeast‐LAB‐AAB cultures at genus, species and intraspecies level.

Significance and Impact of the Study

First study describing microbiological and physicochemical characteristics of palm wine from B. akeassii. Genotypic diversity of palm wine LAB and AAB not reported before is demonstrated and this constitutes valuable information for better understanding of the fermentation which can be used to improve the product quality and develop added value by‐products.     

NITROGEN FIXATION,HYDROGEN CYCLING,AND ELECTRON TRANSPORT KINETICS IN TRICHODESMIUM ERYTHRAEUM (CYANOBACTERIA) STRAIN IMS1011

This study describes the relationships between dinitrogen (N₂) fixation, dihydrogen (H₂) production, and electron transport associated with photosynthesis and respiration in the marine cyanobacterium Trichodesmium erythraeum Ehrenb. strain IMS101. The ratio of H₂ produced:N₂ fixed (H₂:N₂) was controlled by the light intensity and by the light spectral composition and was affected by the growth irradiance level. For Trichodesmium cells grown at 50 μmol photons · m^?2 · s^?1, the rate of N₂ fixation, as measured by acetylene reduction, saturated at light intensities of 200 μmol photons · m^?2 · s^?1. In contrast, net H₂ production continued to increase with light levels up to 1,000 μmol photons · m^?2 · s^?1. The H₂:N₂ ratios increased monotonically with irradiance, and the variable fluorescence measured using a fast repetition rate fluorometer (FRRF) revealed that this increase was accompanied by a progressive reduction of the plastoquinone (PQ) pool. Additions of 2,5‐dibromo‐3‐methyl‐6‐isopropyl‐p‐benzoquinone (DBMIB), an inhibitor of electron transport from PQ pool to PSI, diminished both N₂ fixation and net H₂ production, while the H₂:N₂ ratio increased with increasing level of PQ pool reduction. In the presence of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU), nitrogenase activity declined but could be prolonged by increasing the light intensity and by removing the oxygen supply. These results on the coupling of N₂ fixation and H₂ cycling in Trichodesmium indicate how light intensity and light spectral quality of the open ocean can influence the H₂:N₂ ratio and modulate net H₂ production.     

Electrochemical identification of microbially mediated hydrogen sulfide oxidation

The process of H₂S oxidation by the phototrophic bacteriaThiocapsa roseopersicina andChlorobium phaeobacteroides, respectively, was monitored using a Pt-glass-Ag⁰, Ag₂S electrode combination without liquid junction. Due to the resulting pe(pH) and pH₂S plottings three steps can be distinguished: oxidation of H₂S to an S(0) state, oxidation of S (0) to SO₄ ^2–, and oxidation of the remaining H₂S directly to SO₄ ^2–. Differences between the investigated bacteria exist with respect to their individual oxidation strategies.Thiocapsa apparently stops oxidizing H₂S at pH₂S 7.5 (e.g. 10^–7.5M H₂S) and shifts to the utilization of the intracellularly stored S (0). In contrastChlorobium utilizes its extracellularly stored sulfur parallel to the extracellular H₂S fraction. The corresponding Pt-sensor responses (pe₇ values) were found to be similar to the corresponding partial redox equilibria (p₇ values) of H₂S oxidation stoichiometries as proposed by Van Niel (1931) and Trüper (1964). It is concluded that the recording of pe enables investigators to understand (and control) in situ redox processes, independent of their thermodynamic equilibration, only bound to changes of electroactivity vs. sensor.     

Activation of the l,d‐transpeptidation peptidoglycan cross‐linking pathway by a metallo‐d,d‐carboxypeptidase in Enterococcus faecium

Bypass of the penicillin‐binding proteins by an l ,d ‐transpeptidase (Ldt_fm) confers cross‐resistance to β‐lactam and glycopeptide antibiotics in mutants of Enterococcus faecium selected in vitro. Ldt_fm is produced by the parental strain D344S although it insignificantly contributes to peptidoglycan cross‐linking as pentapeptide stems cannot be used as acyl donors by this enzyme. Here we show that production of the tetrapeptide substrate of Ldt_fm is controlled by a two‐component regulatory system (DdcRS) and a metallo‐d ,d ‐carboxypeptidase (DdcY). The locus was silent in D344S and its activation was due to amino acid substitutions in DdcS or DdcR that led to production of DdcY and hydrolysis of the C‐terminal d ‐Ala residue of the cytoplasmic peptidoglycan precursor UDP‐MurNAc‐pentapeptide. The T¹⁶¹A and T¹⁶¹M substitutions affected a position of DdcS known to be essential for the phosphatase activity of related sensor kinases. Complete elimination of UDP‐MurNAc‐pentapeptide, which was required specifically for resistance to glycopeptides, involved substitutions in DdcY that increased the catalytic efficiency of the enzyme (E¹²⁷K) and affected its interaction with the cell envelope (I¹⁴N). The ddc locus displays striking similarities with portions of the van vancomycin resistance gene clusters, suggesting possible routes of emergence of cross‐resistance to glycopeptides and β‐lactams in natural conditions.     

Characterization and application of a native lactic acid bacterium isolated from tannery fleshings for fermentative bioconversion of tannery fleshings

Lactic acid bacteria (LAB) species isolated from limed and delimed tannery fleshings (TF) were evaluated for their fermentation efficiency and antibacterial property. The native LAB isolates efficiently fermented TF and resulted in a fermented mass with antioxidant properties, indicating their potential for effective eco-friendly bioconversion of TF. From among the LAB isolated, a proteolytic isolate showing better antimicrobial spectrum and reasonably good fermentation efficiency was identified as Enterococcus faecium HAB01 based on various biochemical and molecular tests. This isolate afforded a better degree of hydrolysis (81.36%) of TF than Pediococcus acidilactici (54.64%) that was previously reported by us. The bacteriocin produced by E. faecium was found to be antagonistic to several human pathogens including Listeria, Aeromonas, Staphylococcus and Salmonella. Further, E. faecium HAB01 bacteriocin was thermostable and had a molecular weight of around 5 kDa, apart from being stable at both acidic and alkaline conditions. The bacteriocin was unstable against proteases.     

Inactivation of human pathogens and spoilage bacteria on the surface and internalized within fresh produce by using a combination of ultraviolet light and hydrogen peroxide

Aims: To evaluate the efficacy of ultraviolet (UV) light (254 nm) combined with hydrogen peroxide (H₂O₂) to inactivate bacteria on and within fresh produce. Methods and Results: The produce was steep inoculated in bacterial cell suspension followed by vacuum infiltration. The inoculated samples were sprayed with H₂O₂ under constant UV illumination. The log count reduction (LCR) of Salmonella on and within lettuce was dependent on the H₂O₂ concentration, temperature and treatment time with UV intensity being less significant. By using the optimized parameters (1·5% H₂O₂ at 50°C, UV dose of 37·8 mJ cm^?2), the surface Salmonella were reduced by 4·12 ± 0·45 and internal counts by 2·84 ± 0·34 log CFU, which was significantly higher compared with H₂O₂ or UV alone. Higher LCR of Escherichia coli O157:H7, Pectobacterium carotovora, Pseudomonas fluorescens and Salmonella were achieved on leafy vegetables compared with produce, such as cauliflower. In all cases, the surface LCR were significantly higher compared with the samples treated with 200 ppm hypochlorite. UV–H₂O₂‐treated lettuce did not develop brown discolouration during storage but growth of residual survivors occurred with samples held at 25°C. Conclusions: UV–H₂O₂ reduce the bacterial populations on and within fresh produce without affecting the shelf‐life stability. Significance of the Study: UV–H₂O₂ represent an alternative to hypochlorite washes to decontaminate fresh produce.