Effects of growth conditions on the activities of superoxide dismutase and NADH-oxidase/NADH-peroxidase inStreptococcus lactis

An increase in the superoxide dismutase (SOD) activity inStreptococcus lactis was observed when the cells were grown at increased oxygen partial pressures or exposed to hyperbaric oxygen tensions. The NADH-oxidase/NADH-peroxidase activities inS. lactis increased in galactose-grown cells when cultivated in air compared with N₂/CO₂. This effect did not occur when glucose was the carbon source; however, an increase in the activities of these enzymes was observed in oxygen atmosphere. The correlation between SOD, NADH-oxidase/NADH-peroxidase, and the metabolic pathways involved is discussed. The effect of manganese on the SOD activity is also considered.     

Improvement of the respiration efficiency of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> by decreasing the culture pH

Objective

The growth characteristics and intracellular hemin concentrations of Lactococcus lactis grown under different culture pH and aeration conditions were examined to investigate the effect of culture pH on the respiration efficiency of L. lactis NZ9000 (pZN8148).

Results

Cell biomass and biomass yield of L. lactis grown with 4 μg hemin/ml and O₂ were higher than those without aeration when the culture pH was controlled at 5–6.5. The culture pH affected the respiratory efficiency in the following order of pH: 5 > 5.5 > 6 > 6.5; the lag phase increased as the culture pH decreased. Hemin accumulation was sensitive to culture pH. Among the four pH conditions, pH 5.5 was optimal for hemin accumulation in the cells. The highest intracellular hemin level in L. lactis resting cells incubated at different pH saline levels (5–6.5) was at pH 5.5.

Conclusion

The respiration efficiency of L. lactis under respiration-permissive conditions increases markedly as the culture pH decreases. These results may help develop high cell-density L. lactis cultures. Thus, this microorganism may be used for industrial applications.

     

Inhibition of Cyanide-Insensitive Respiration in <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> SYSU-011 by 8-Hydroxyquinolone

The inhibition of the cyanide (KCN)-insensitive respiration of Klebsiella oxytoca SYSU-011 by 8-hydroxyquinoline (8-HQ) was determined. Results showed that the profile of the rate of oxygen uptake of normal-grown and 8-HQ–grown K. oxytoca SYSU-011 was biphasic and similar, suggesting that 8-HQ did not inhibit the respiration of normal-grown K. oxytoca SYSU-011. A different biphasic KCN inhibition profile of respiration was observed for KCN-grown cells treated with and without 8-HQ. No decrease in respiration rate of KCN-grown cells and a 40% decrease in respiration rate of KCN-grown cells treated with 8-HQ were observed when KCN concentration was 10^–1 mM. Comparing differences of the profiles of oxygen uptake in KCN-grown cells with and without 8-HQ addition indicated that 8-HQ inhibited expression of the KCN-insensitive pathway carried out by nonheme oxidase. Greater inhibition of NADH oxidase activity by 2-n-heptyl-4-hydroxyquinoline-N-oxide from the cell membrane of the KCN-grown cells treated with 8-HQ, and more H₂O₂ production from these cells with than without 8-HQ, suggest that the function of the cyanide-insensitive pathway can stabilize the respiration of the cyanide-grown cells to prevent the production of H₂O₂.     

Nisin production of Lactococcus lactis N8 with hemin‐stimulated cell respiration in fed‐batch fermentation system

In this study, nisin production of Lactococcus lactis N8 was optimized by independent variables of glucose, hemin and oxygen concentrations in fed‐batch fermentation in which respiration of cells was stimulated with hemin. Response surface model was able to explain the changes of the nisin production of L. lactis N8 in fed‐batch fermentation system with high fidelity (R² 98%) and insignificant lack of fit. Accordingly, the equation developed indicated the optimum parameters for glucose, hemin, and dissolved oxygen were 8 g L^?1 h^?1, 3 μg mL^?1 and 40%, respectively. While 1711 IU mL^?1 nisin was produced by L. lactis N8 in control fed‐batch fermentation, 5410 IU mL^?1 nisin production was achieved within the relevant optimum parameters where the respiration of cell was stimulated with hemin. Accordingly, nisin production was enhanced 3.1 fold in fed‐batch fermentation using hemin. In conclusion the nisin production of L. lactis N8 was enhanced extensively as a result of increasing the biomass by stimulating the cell respiration with adding the hemin in the fed‐batch fermentation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:678–685, 2015     

Denitrification of nitrate to nitrogen gas by washed cells ofRhizobium japonicum and by bacteroids fromGlycine max

Nitrate, nitrite and nitrous oxide were denitrified to N₂ gas by washed cells ofRhizobium japonicum CC706 as well as by bacteroids prepared from root nodules ofGlycine max (L.) Merr. (CV. Clark 63). Radiolabelled N₂ was produced from either K¹⁵NO₃ or Na¹⁵NO₂ by washed cells ofRh. japonicum CC705 grown with either nitrate only (5 mM) or nitrate (5 mM) plus glutamate (10 mM). Nitrogen gas was also produced from N₂O. Similar results were obtained with bacteroids ofG. max. The stoichiometry for the utilization of¹⁵NO ₃ ^- or¹⁵NO ₂ ^- and the produciton of¹⁵N₂ was 2:1 and for N₂O utilization and N₂ production it was 1:1. Some of the¹⁵N₂ gas produced by denitrification of¹⁵NO ₃ ^- in bacteroids was recycled via nitrogenase into cell nitrogen.     

Acetoin Fermentation by Citrate-Positive Lactococcus lactis subsp. lactis 3022 Grown Aerobically in the Presence of Hemin or Cu2+

Citr⁺Lactococcus lactis subsp. lactis 3022 produced more biomass and converted most of the glucose substrate to diacetyl and acetoin when grown aerobically with hemin and Cu²⁺. The activity of diacetyl synthase was greatly stimulated by the addition of hemin or Cu²⁺, and the activity of NAD-dependent diacetyl reductase was very high. Hemin did not affect the activities of NADH oxidase and lactate dehydrogenase. These results indicated that the pyruvate formed via glycolysis would be rapidly converted to diacetyl and that the diacetyl would then be converted to acetoin by the NAD-dependent diacetyl reductase to reoxidize NADH when the cells were grown aerobically with hemin or Cu²⁺. On the other hand, the Y_Glu value for the hemincontaining culture was lower than for the culture without hemin, because acetate production was repressed when an excess of glucose was present. However, in the presence of lipoic acid, an essential cofactor of the dihydrolipoamide acetyltransferase part of the pyruvate dehydrogenase complex, hemin or Cu²⁺ enhanced acetate production and then repressed diacetyl and acetoin production. The activity of diacetyl synthase was lowered by the addition of lipoic acid. These results indicate that hemin or Cu²⁺ stimulates acetyl coenzyme A (acetyl-CoA) formation from pyruvate and that lipoic acid inhibits the condensation of acetyl-CoA with hydroxyethylthiamine PP_i. In addition, it appears that acetyl-CoA not used for diacetyl synthesis is converted to acetate.     

Balance between Cyanide-Sensitive and -Insensitive Respiration in Wheat Root Mitochondria, as Influenced by Salt Concentration in the Plant Growth Medium

Mitochondria were isolated from 7-day-old wheat roots (Triticum vulgare Vill. cv. Svenno Spring Wheat) grown in either a full-strength culture medium (100%) or in the same medium diluted 100 times (1%). Outer membrane integrity was assayed using the cytochrome c reduction assay. This indicated about 20% damage. Using an oxygen electrode the respiration of the mitochondria was measured with either malate or succinate as the substrate (both 40 mM). KCN (3 mM) and salicylhydroxamic acid (SHAM, 1 mM) were used as inhibitors. The properties of the isolated mitochondria (STATE 3 rate, ADP/O ratio, and KCN-sensitivity) depend upon the ionic concentration of the growth medium of the roots. In the mitochondria isolated from roots grown in the 1% medium (1% mitochondria) there is a synergistic effect of KCN and SHAM. This means that electrons can be shifted from one pathway to the other when only one of the inhibitors is added. This flexibility between the electron pathways is almost nil in the mitochondria isolated from roots grown in the 100% medium (100% mitochondria). The maximal capacity of the alternative electron pathway (= rate in the presence of KCN) is higher in 1% (40 nmol O₂ min^?1 (mg protein)^?1) than in 100% mitochondria (20 nmol O₂ min^?1 (mg protein)^?1. In 100% mitochondria the alternative pathway seems to be operating at maximal capacity in the absence of KCN with both substrates and in both STATES 3 and 4. In 1% mitochondria the alternative pathway functions at >50% of its capacity in the absence of KCN.     

Alternative respiration and antibiotic production of Acremonium chrysogenum

The influence of potassium cyanide (KCN), dissolved O₂ concentration and medium composition on alternative respiration (AR) of Acremonium chrysogenum were investigated. The respiration of the fungus was only partially inhibited by KCN, but completely inhibited by the combination of KCN with salicylhydroxamic acid. It has been proved by in-situ measurements of the NADH-dependent fluorescence that the AR is active at low dissolved O₂ concentrations. The influence of the medium composition and the age of the fungus on the specific oxygen uptake rate is considered. Correpondence to: K. Schügerl     

Aerobic Respiration of Kappa Particles from Paramecium aurelia,Stock 51*

SYNOPSIS. Kappa particles from killer cultures of stock 51 Paramecium aurelia were purified and their respiration measured polarographically. The slight bacterial contaminations in the kappa preparations were not significant. Freshly collected kappa in dilute buffer at room temperature had an endogenous Q_O2 of 17.0 ± 1.6 μl/mg dry weight/hr (mean ± standard error). The Q_O2 decayed 50% in 5 hr. Among the sugars tested only glucose and sucrose increased the respiratory rate of kappa. The di- and tri-carboxylates of the tricarboxylic acid cycle stimulated the respiration of kappa. KCN, CO and 2-heptyl-4-hydroxyquinoline N-oxide (HOQNO) inhibited respiration. These findings ensure an organismic status for kappa and justify the belief that it is bacterial in origin.     

Lactococcus lactis as a Cell Factory for High-Level Diacetyl Production

We report the engineering of Lactococcus lactis for the efficient conversion of sugar into diacetyl by combining NADH-oxidase overproduction and α-acetolactate decarboxylase inactivation. Eighty percent of the carbon flux was found to be rerouted via α-acetolactate to the production of diacetyl by preloading the cells with NADH-oxidase before their use as a cell factory.     

Growth inhibition of bacteria by tributylgermanium acetate and its reversal by blood constituents

Growth inhibition ofMycobacterium phlei by tributylgermanium acetate can be reversed by addition of large amounts of blood or blood serum to the nutrient medium; the blood erythrocyte fraction is inactive.On the other hand growth inhibition of the lactic acid bacteriaStreptococcus lactis andLeuconostoc mesenteroides can be counteracted by traces of blood or its crythrocyte fraction, by hemin and by catalase. Blood serum even in large amounts is ineffective.Whereas tributylgermanium acetate is highly active against most lactic acid bacteria studied, the antagonistic action of blood or hemin is found only with the species named. The possible mechanism of this antagonism is discussed.     

Toxicity of organotins towards the marine yeastDebaryomyces hansenii

Of nine organotin compounds tested towards the marine yeastDebaryomyces hansenii, only triphenyltin chloride (Ph₃SnCl) and mono-, di-, and tributyltin chloride induced significant K⁺ release from cells which was symptomatic of viability loss. The general order of toxicity of the butylated compounds was tributyltin chloride (Bu₃SnCl) > monobutyltin chloride (BuSnCl₃) dibutyltin chloride (Bu₂SnCl₂). The overall toxicity of Ph₃SnCl was similar to BuSnCl₃. Release of K⁺ induced by butylated tin compounds or by Ph₃SnCl was strongly dependent on the external pH. Maximal toxicity occurred at pH 6.5 for Bu₃SnCl, BuSnCl₃, and Ph₃SnCl, whereas maximal toxicity of Bu₂SnCl₂ occurred at pH 5.0. Toxicity was decreased above or below these values. The toxicity of BuSnCl₃, Bu₃SnCl, and Ph₃SnCl was reduced at salinity levels approximating to sea water conditions. Prior growth ofD. hansenii in 3% (w/v) NaCl also resulted in reduced sensitivity to Bu₃SnCl as evidenced by a decreased rate and extent of K⁺ efflux. Bu₃SnCl-induced Na⁺ release from cells grown in the absence or presence of 3% (w/v) NaCl was low and similar in both cases. It appeared that the monovalent cation was important in the reduction of Bu₃SnCl toxicity since Na₂SO₄ had a similar protective effect as NaCl while CsCl completely prevented K⁺ efflux. Thus, the effects of external NaCl were related both to Na⁺ and to Cl^–. These results emphasize that cellular and environmental factors influence the toxic effects of organotins and suggests that these compounds may be more effective antimicrobial agents in some environmental niches than in others.     

Branched respiratory chain in aerobically grown Staphylococcus aureus —oxidation of ethanol by cells and protoplasts

Addition of ethanol and some other primary alcohols, except methanol, to cells and protoplasts (but not membrane particles) considerably stimulated the rate of oxygen consumption. This additional respiration was strongly inhibited by 0.1 mM KCN. The cyanide inhibition curve of endogenous substrate oxidation was slightly biphasic while in the presence of ethanol it became clearly biphasic having K _i values of approx. 0.1 and 0.5 mM. Based on the steady-state cytochrome spectra in the presence of 0.1 mM KCN, we attributed the lower K _i to cytochrome a ₆₀₂. Proteolysis of protoplasts external membrane proteins did not change the rate of endogeneous substrate oxidation but prevented the inhibition of this respiration by low concentrations of KCN and stimulation of oxygen consumption by ethanol. The activity of NAD⁺-dependent ethanol dehydrogenase in the cytoplasm was found to be 520 nmol NADH-x min^–1 x mg^–1 protein. Proteolysis of external membrane proteins apparently inhibits the operation of the cytochrome a ₆₀₂-containing electron transport branch inducing the suppression of electron flow from NADH to oxygen.     

Interspecies sex-specific growth responses inKluyveromyces

The sex-specific growth factors produced by thea and mating types ofKluyveromyces lactis were examined for interspecies cross-reactivity on growing cells ofKluyveromyces aestuarii andKluyveromyces delphensis. Taxonomically,K. aestuarii is closely related toK. lactis;K. delphensis is more distantly related. Reciprocal growth responses occurred betweenK. aestuarii and the a mating type ofK. lactis; no response was evident betweenK. delphensis and either sex ofK. lactis.     

Transport of hemin byHaemophilus influenzae type b

Haemophilus influenzae may be distinguished from other gram-negative bacteria by its growth requirement for hemin. The ability of this bacterium to accumulate hemin while growing in a fully defined medium has been partially characterized.Haemophilus influenzae type b ATCC 9795 transported hemin at a rate of 1.2 pmol/min/10⁹ cells during logarithmic growth. The kinetics of active transport of doubly radiolabeled hemin indicated that both iron and the porphyrin ring were taken up at the same rate. Hemin satisfied some of the total iron requirement ofHaemophilus as determined by starving the cells for iron with the addition of ethylenediamine-di-(o-hydroxyphenylacetic acid) (EDDA) and by limiting the porphyrin supply. Outer membrane proteins were compared from cells grown under hemin sufficiency versus cells grown under hemin starvation: in the latter case, a protein of molecular weight 43,000 was present in enhanced amounts; this protein may play a role in the permeability of hemin across the cell envelope ofH. influenzae type b.     

Inhibition of specific electron transport pathways leads to oxidative stress and decreased Candida albicans proliferation

Candida parapsilosis mitochondria contain three respiratory chains: the classical respiratory chain (CRC), a secondary parallel chain (PAR) and an “alternative” oxidative pathway (AOX). We report here the existence of similar pathways in C. albicans. To observe the capacity of each pathway to sustain yeast growth, C. albicans cells were cultured in the presence of inhibitors of these pathways. Antimycin A and KCN totally abrogated yeast growth, while rotenone did not prevent proliferation. Furthermore, rotenone promoted only partial respiratory inhibition. Lower concentrations of KCN that promote partial inhibition of respiration did not inhibit yeast growth, while partial inhibition of respiration with antimycin A did. Similarly, AOX inhibitor BHAM decreased O₂ consumption slightly but completely stunted cell growth. Reactive oxygen species production and oxidized glutathione levels were enhanced in cells treated with antimycin A or BHAM, but not rotenone or KCN. These findings suggest that oxidative stress prevents C. albicans growth.     

A food-grade delivery system for<Emphasis Type="Italic"> Lactococcus lactis</Emphasis> and evaluation of inducible gene expression

The genetic improvement of Lactococcus lactis is a matter of biotechnological interest in the food industry and in the pharmaceutical and medical fields. However, to construct a food-grade delivery system, both the presence of antibiotic markers or plasmid sequences should be avoided and the maintenance and expression of the cloned gene should be guaranteed. The objective of this work was to produce crossover mutants of L. lactis with a reporter gene under the control of an inducible promoter in order to evaluate the level of gene expression. We utilized a nuclease gene of Staphylococcus aureus as a reporter gene, P _nisA as the nisin-inducible promoter, a non-essential gene involved in histidine biosynthesis of L. lactis as the site for homologous recombination, and pRV300 as a suicide vector for the genomic integration in L. lactis NZ9000. Single- and double-crossover mutants were identified by genotype and phenotype. Relative to episomal transformants of L. lactis, the level of expression of the heterologous protein after nisin induction was similar in the crossover mutants, suggesting that a single copy of the heterologous gene can be used to produce the protein of interest.     

Optimization of fermentation conditions for the expression of sweet-tasting protein brazzein in Lactococcus lactis

Aims: To improve the production of sweet‐tasting protein brazzein in Lactococcus lactis using controlled fermentation conditions. Methods and Results: The nisin‐controlled expression system was used for brazzein expression. The concentration of nisin for induction and the optical density (OD) at induction were therefore optimized, together with growth conditions (medium composition, pH, aerobic growth in the presence of hemin). Brazzein was assayed with ELISA on Ni‐NTA plates and Western blot. Use of the M‐17 medium, containing 2·5% glucose, anaerobic growth at pH 5·9 and induction with 40 ng ml^?1 nisin at OD 3·0 led to an approx. 17‐fold increase in brazzein per cell production compared to non‐optimized starting conditions. Aerobic growth in the presence of hemin did not increase the production. Conclusions: Considerable increase in brazzein per cell production was obtained at optimized fermentation conditions. Significance and Impact of the Study: Optimized growth conditions could be used in application of brazzein expression in L. lactis. The importance of pH and OD at induction contributes to the body of knowledge of optimal recombinant protein expression in L. lactis. The new assay for brazzein quantification was introduced.     

Cytochrome d expression and regulation pattern in free-living Rhizobium phaseoli

The respiratory system of Rhizobium phaseoli CFN42 in free-living cultures was studied. Cytochromes b, c, o and aa ₃ were found in fast growing cells cultured under forced aeration. Stationary aerobic cells, and semianaerobically grown cells showed decreased levels of cytochromes c, aa ₃ and o, concomitant with a significant increase of b type cytochromes and the synthesis of a new cytochrome, tentatively identified as cytochrome d. Cell membranes with the highest content of cytochrome d (semianaerobically grown cells) showed the highest respiratory activities with NADH, succinate, malate or ascorbate-TMPD (N,N,N,N-tetramethyl p-phenylendiamine). In the presence of either of the above electron donors, cytochrome d was clearly reduced. NADH dependent respiration in membranes of fast growing cells (no cytochrome d detected) was abolished by 25 M KCN. This inhibitor concentration caused only 15–20% inhibition in membranes of semianaerobically grown cells (cyt d present). Moreover, in the presence of 1–5 mM KCN, the oxidation of cyt d and a b type cytochromes was spectrally detected. It is suggested that cyt d is a functional cytochrome in the respiratory system of free-living Rhizobia, probably acting as terminal oxidase.     

Experimental bioenergetics ofSaccharomyces cerevisiae in respiration and fermentation

Aerobic growth of Saccharomyces cerevisiae on glucose was investigated, focusing on the heat evolution as it relates to biomass and ethanol synthesis. “Aerobic fermentation” and “aerobic respiration” were established respectively in the experimental system by performing batch and fed-batch experiments. “Balanced growth” batch cultivations were carried out with initial sugar concentrations ranging from 10 to 70 g/L, resulting in different degrees of catabolite repression. The fermentative heat generation was continuously monitored in addition to the key culture parameters such as ethanol production rate, CO₂ evolution rate, O₂ uptake rate, specific growth rate, and sugar consumption rate. The respective variations of the above quantities reflecting the variations in the catabolic activity of the culture were studied. This was done in order to evaluate the microbial regulatory system, the energetics of microbial growth including the rate of heat evolution and the distribution of organic substrate between respiration and fermentation. This study was supported by closing C, energy, and electron balances on the system. The comparison of the fractions of substrate energy evolved as heat (δ_h) with the fraction of available electrons transferred to oxygen (?_O2) indicated equal values of the two (0.46) in the aerobic respiration (fed-batch cultivation). However, the glucose effect in batch cultivations resulted in smaller ?_O2 than δ_h, while both values decreased in their absolute values. The evaluation of the heat energetic yield coefficients, together with the fraction of the available electrons transferred to O, contributed to the estimation of the extent of heat production through oxidative phosphorylation.