Location of tyrosine phenol-lyase in some Gram-negative bacteria

Abstract From various habitats (plant material, fruits, soil), yeasts belonging to the species of Pichia kluyveri and Hanseniaspora uvarum were isolated that showed killer activity. According to the activity spectrum against other yeasts these strains belonged to 11 different groups that were distinguishable from the killer strains K₁-K₁₀. The isoelectric points of the killer proteins were in the range of pH 3.5–3.9, the activity optimum was observed at pH 4.2–4.6. Above pH 5 and above a temperature of 25–35°C the killer proteins were inactivated.     

Signaling networks controlling mucin production in response to Gram-positive and Gram-negative bacteria

Human lung cells exposed to pathogenic bacteria upregulate the production of mucin, the major macromolecular component of mucus. Generally this upregulation is beneficial for the host, however, in the lungs of cystic fibrosis patients, overproduction of mucin can lead to the plugging of pulmonary airways. Mucus plugging impedes airflow and creates an environment that is highly compartmentalized: those bacteria within the mucus layer are shielded from high doses of antibiotics whereas those outside the mucus are exposed. These conditions augment mutation rate and the development of drug resistance in bacteria that colonize the lungs of cystic fibrosis patients. While therapeutic inhibition of mucin induction would improve airflow and reduce antibiotic resistance in these patients, the challenge is to develop drugs that block excessive mucin production while leaving beneficial aspects of the response intact. To do this, we must understand the molecular mechanisms underlying mucin production. Here we review the signal transduction pathways that control mucin production in response to Gram-positive and Gram-negative bacteria.     

Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase.

Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The ldh mutation was found to map at 30.5 min on the E. coli chromosome. The ldh mutant FMJ39 showed no detectable lactate dehydrogenase activity and produced no lactic acid from glucose under anaerobic conditions as estimated by in vivo nuclear magnetic resonance measurements. We also found that in wild-type strains the fermentative lactate dehydrogenase was conjointly induced by anaerobic conditions and an acidic pH. Despite previous findings that phosphate concentrations affect the proportion of lactic acid produced during fermentation, we were unable to find any intrinsic effect of phosphate on lactate dehydrogenase activity, apart from the buffering effect of this ion.     

Kinetics and thermodynamics of lactate dehydrogenases from beef heart, beef muscle, and flounder muscle.

The eight rate constants for a four-step ordered ternary-complex mechanism have been compared for lactate dehydrogenases (EC1.1.1.27) from three sources, beef heart, beef muscle, and flounder muscle. The rate constants were determined at temperatures ranging from 5 degrees C to 50 degrees C, and the corresponding activation parameters deltaG not equal to, deltaH not equal to, and deltaS not equal to were calculated. Significant differences are noted for the values for the three types of enzyme. The relative heights of the activation barriers are much the same in all three cases, differences in kinetic behavior resulting mainly from differences in the stable binary and ternary enzyme-substrate complexes. These complexes are, in general, at lower free-energy and enthalpy levels of the beef-heart and beef-muscle enzymes than for the flounder-muscle enzyme. A high degree of compensation is found between the enthalpies and entropies of activation, resulting in relatively small differences between the free energies (and rates) for homologous steps with different enzymes. Analysis of the results, on the assumption that the compensation effect is due to weak-bonding effects, suggests that there are fewer weak bonds in the stable complexes of the muscle enzymes.     

Tolerance to acid in pH 5.0-grown organisms of potentially pathogenic Gram-negative bacteria

S.A. NOJOUMI, D.G. SMITH AND R.J. ROWBURY. 1995. A wide range of potentially pathogenic species of Gram-negative bacteria were far more resistant to extreme acidity (pH 2.0–3.5) when cultured at pH 5.0 (habituated to acid) than after pH 7.0 culture. The differences were particularly great for Citrobacter spp., Enterobacter spp., Klebsiella spp. and for Vibrio parahaemolyticus ; substantial habituation was also observed for Proteus mirabilis and Aeromonas formicans but the effect was less marked for Serratia marcescens and Acinetobacter calcoaceticus . Growth at pH 5.0 was substantially poorer than at pH 7.0 for most of the above species and also for Salmonella typhimurium and Salm. enteritidis but phosphate markedly enhanced growth at pH 5.0 for many of these species without affecting growth at pH 7.0.     

Effect of pH on growth rates of rumen amylolytic and lactilytic bacteria.

The relationship between the pH of the medium and specific growth rates, in well-buffered media at 38.5 degrees C, was determined for three strains of Butyrivibrio fibrisolvens and for one strain each of Streptococcus bovis, Selenomonas ruminantium subsp. lactilytica. Megasphaera elsdenii, Veillonella alcalescens, and Propionibacterium acnes. The pH optima for growth were between 6.1 and 6.6 for all six species, and the upper pH limits were between 7.3 and 7.8. The lower limit pH values for growth on glucose were 5.4 for B. fibrisolvens, near 5.0 for V. alcalescens, and between 4.4 and 4.8 for the other four species. These values fall within the minimum pH ranges found when these species are grown in poorly buffered medium with nonlimiting glucose concentrations. Acid sensitivity per se could cause the washout of B. fibrisolvens, but not of the other five species, from the rumens of animals on high-starch diets.     

On the possible roles of N-terminal His-rich domains of Cu,Zn SODs of some Gram-negative bacteria

The Cu,Zn superoxide dismutases (Cu,Zn SOD) isolated from some Gram-negative bacteria possess a His-rich N-terminal metal binding extension. The N-terminal domain of Haemophilus ducreyi Cu,Zn SOD has been previously proposed to play a copper(II)-, and may be a zinc(II)-chaperoning role under metal ion starvation, and to behave as a temporary (low activity) superoxide dismutating center if copper(II) is available. The N-terminal extension of Cu,Zn SOD from Actinobacillus pleuropneumoniae starts with an analogous sequence (HxDHxH), but contains considerably fewer metal binding sites. In order to study the possibility of the generalization of the above mentioned functions over all Gram-negative bacteria possessing His-rich N-terminal extension, here we report thermodynamic and solution structural analysis of the copper(II) and zinc(II) complexes of a peptide corresponding to the first eight amino acids (HADHDHKK-NH₂, L) of the enzyme isolated from A. pleuropneumoniae. In equimolar solutions of Cu(II)/Zn(II) and the peptide the MH₂L complexes are dominant in the neutral pH-range. L has extraordinary copper(II) sequestering capacity (K_D,Cu = 7.4 × 10^− 13 M at pH 7.4), which is provided only by non-amide (side chain) donors. The central ion in CuH₂L is coordinated by four nitrogens {NH₂,3N_im} in the equatorial plane. In ZnH₂L the peptide binds to zinc(II) through a {NH₂,2N_im,COO⁻} donor set, and its zinc binding affinity is relatively modest (K_D,Zn = 4.8 × 10^− 7 M at pH 7.4). Consequently, the presented data do support a general chaperoning role of the N-terminal His-rich region of Gram-negative bacteria in copper(II) uptake, but do not confirm similar function for zinc(II). Interestingly, the complex CuH₂L has very high SOD-like activity, which may further support the multifunctional role of the copper(II)-bound N-terminal His-rich domain of Cu,Zn SODs of Gram-negative bacteria. The proposed structure for the MH₂L complexes has been verified by semiempirical quantum chemical calculations (PM6), too.     

Ultrastructure of bacteria and the proportion of Gram-negative bacteria in marine sediments

Bacteria in sediments from the surface aerobic layer (0–1 cm) and a deeper anaerobic layer (20–21 cm) of a seagrass bed were examined in section by transmission electron microscopy. Bacteria with a Gram-negative ultrastructure made up 90% of bacteria in the surface layer, and Gram-positive bacteria comprised 10%. In the anaerobic zone, Gram-negative bacteria comprised 70% and Gram-positive bacteria 30% of the bacterial population. These differences were highly significant and support predictions of these proportions made from muramic acid measurements and direct counting with fluorescence microscopy. Most cells were enveloped in extracellular slime layers or envelopes, some with considerable structural complexity. The trophic value to animals of these envelopes is discussed. A unique organism with spines was observed.     

Nisin, temperature and pH effects on growth and viability of Pectinatus frisingensis, a Gram-negative, strictly anaerobic beer-spoilage bacterium

Pectinatus frisingensis , a Gram-negative and strictly anaerobic beer spoilage bacterium is sensitive to nisin. An increase in nisin concentration (0 to 1100 IU ml⁻¹) added to the culture medium prolonged the lag phase, and decreased the growth rate of the bacterium. In addition, late exponential cells of P. frisingensis exposed to low concentrations of nisin lost immediately a part of their intracellular K⁺. Presence of Mg²⁺ up to 15 mmol l⁻¹ did not protect P. frisingensis from nisin-induced loss of viability and K⁺ efflux. Potassium leaks were also measured in P. frisingensis late exponential phase cells exposed to combined effects of nisin addition (100–500 IU ml⁻¹), 10 min mild heat-treatment (50 °C) or rapid cooling (2 °C), and pH (4·0 and 6·2). Net K⁺ efflux from both starving and glucose-metabolizing cells, was more important at pH 6·2, whatever the temperature treatment and nisin addition. Reincubation at 30 °C of P. frisingensis glucose-metabolizing cells exposed to a preliminary combination of nisin addition and mild heat or cooling down treatment, showed that cells exposed to rapid cooling reaccumulated more K₊ than heat-treated cells, whatever the pH conditions. A combination of nisin and mild heat-treatment could thus be of interest to prevent P. frisingensis growth in beers.     

Distribution of the phosphoenolpyruvate:glucose phosphotransferase system in fermentative bacteria.

A number of selected fermentative bacteria were surveyed for the presence of the phosphoenolpyruvate:glucose phosphotransferase system, with particular attention to those organisms which ferment glucose by pathways other than the Embden-Meyerhof-Parnas pathway. The phosphoenolpyruvate:glusoe phosphotransferase system was found in all homofermentative lactic acid bacteria tested that ferment glucose via the Embden-Meyerhof-Parnas pathway, but in none of a group of heterofermentative species of Lactobacillus or Leuconostoc, which ferment glucose via the phosphoketolase pathway. A phosphoenolpyruvate:glucose phosphotransferase system was also absent in Zymomonas mobilis, which ferments glucose via an anaerobic Entner-Doudoroff pathway. It thus appears that the phosphotransferase mode of glucose transport is limited to bacteria with the Embden-Meyerhof-Parnas mode of glucose fermentation.     

Pure-culture growth of fermentative bacteria, facilitated by H2 removal: bioenergetics and H2 production

We used an H2-purging culture vessel to replace an H2-consuming syntrophic partner, allowing the growth of pure cultures of Syntrophothermus lipocalidus on butyrate and Aminobacterium colombiense on alanine. By decoupling the syntrophic association, it was possible to manipulate and monitor the single organism's growth environment and determine the change in Gibbs free energy yield (DeltaG) in response to changes in the concentrations of reactants and products, the purging rate, and the temperature. In each of these situations, H2 production changed such that DeltaG remained nearly constant for each organism (-11.1 +/- 1.4 kJ mol butyrate(-1) for S. lipocalidus and -58.2 +/- 1.0 kJ mol alanine(-1) for A. colombiense). The cellular maintenance energy, determined from the DeltaG value and the hydrogen production rate at the point where the cell number was constant, was 4.6 x 10(-13) kJ cell(-1) day(-1) for S. lipocalidus at 55 degrees C and 6.2 x 10(-13) kJ cell(-1) day(-1) for A. colombiense at 37 degrees C. S. lipocalidus, in particular, seems adapted to thrive under conditions of low energy availability.     

Determining the impacts of fermentative bacteria on wollastonite dissolution kinetics

Silicate minerals can be a source of calcium and alkalinity, enabling CO₂ sequestration in the form of carbonates. For this to occur, the mineral needs to be first dissolved in an acidifying process such as the biological process of anaerobic fermentation. In the present study, the main factors which govern the dissolution process of an alkaline silicate mineral (wollastonite, CaSiO₃) in an anaerobic fermentation process were determined. Wollastonite dissolution kinetics was measured in a series of chemical batch experiments in order to be able to estimate the required amount of alkaline silicate that can neutralize the acidifying fermentation process. An anaerobic fermentation of glucose with wollastonite as the neutralizing agent was consequently performed in a fed-batch reactor. Results of this experiment were compared with an abiotic (control) fed-batch reactor in which the fermentation products (i.e. organic acids and alcohols) were externally supplied to the system at comparable rates and proportions, in order to provide chemical conditions similar to those during the biotic (fermentation) experiment. This procedure enabled us to determine whether dissolution of wollastonite was solely enhanced by production of organic acids or whether there were other impacts that fermentative bacteria could have on the mineral dissolution rate. The established pH profiles, which were the direct indicator of the dissolution rate, were comparable in both experiments suggesting that the mineral dissolution rate was mostly influenced by the quantity of the organic acids produced.     

Phospholipid composition and cardiolipin synthesis in fermentative and nonfermentative marine bacteria.

Twenty biochemically distinct isolates of marine bacteria, comprising a collection of gram-negative, motile, straight and curved rod-shaped organisms, were separated into fermentative and nonfermentative groups. The isolates were analyzed fro phospholipid composition and the activities of the enzymes, cardiolipin synthetase, and a phosphilipase were determined. The phospholipid compositions of all isolates were generally similar. Phosphatidylethanolamine and phosphatidylglycerol were the major phospholipid classes detected. The absence of cardiolipin in most of the nonfermentative isolates was the most striking observation noted. This was verified chromatographically and by the absence of cardiolipin synthetase activity. In isolates which had cardiolipin, it apparently was synthesized by the condensation of two molecules of phosphatidylglycerol, a mechanism similar to that observed in terrestrial bacteria. Possible correlations between the presence of cardiolipin and Mg-2+ requirements for growth are discussed.     

Characterization of radiation-resistant vegetative bacteria in beef.

Ground beef contains numerous microorganisms of various types. The commonly recognized bacteria are associated with current problems of spoilage. Irradiation, however, contributes a new factor through selective destruction of the microflora. The residual microorganisms surviving a nonsterilizing dose are predominantly gram-negative coccobacilli. Various classifications have been given, e.g., Moraxella, Acinetobacter, Achromobacter, etc. For a more detailed study of these radiation-resistant bacteria occurring in ground beef, an enrichment procedure was used for isolation. By means of morphological and biochemical tests, most of the isolates were found to be Moraxella, based on current classifications. The range of growth temperatures was from 2 to 50 C. These bacteria were relatively heat sensitive, e.g., D10 of 5.4 min at 70 C or less. The radiation resistance ranged from D10 values of 273 to 2,039 krad. Thus, some were more resistant than any presently recognized spores. A reference culture of Moraxella osloensis was irradiated under conditions comparable to the enrichment procedure used with the ground beef. The only apparent changes were in morphology and penicillin sensitivity. However, after a few subcultures these bacteria reverted to the characteristics of the parent strain. Thus, it is apparent that these isolates are a part of the normal flora of ground beef and not aberrant forms arising from the irradiation procedure. The significance, if any, of these bacteria is not presently recognized.     

Sensitivity of some marine bacteria, a moderate halophile, and Escherichia coli to uncouplers at alkaline pH.

The inhibitory effects of uncouplers on amino acid transport into three marine bacteria, Vibrio alginolyticus 118, Vibrio parahaemolyticus 113, and Alteromonas haloplanktis 214, into a moderate halophile, Vibrio costicola NRC 37001, and into Escherichia coli K-12 were found to vary depending upon the uncoupler tested, its concentration, and the pH. Higher concentrations of all of the uncouplers were required to inhibit transport at pH 8.5 than at pH 7.0. The protonophore carbonyl cyanide m-chlorophenylhydrazone showed the greatest reduction in inhibitory capacity as the pH was increased, carbonyl cyanide p-trifluoromethoxyphenylhydrazone showed less reduction, and 3,3',4',5-tetrachlorosalicylanilide was almost as effective as an inhibitor of amino acid transport at pH 8.5 as at pH 7.0 for all of the organisms except A. haloplanktis 214. Differences between the protonophores in their relative activities at pHs 7.0 and 8.5 were attributed to differences in their pK values. 3,3',4',5-Tetrachlorosalicylanilide, carbonyl cyanide m-chlorophenylhydrazone, 2-heptyl-4-hydroxyquinoline-N-oxide, and NaCN all inhibited Na+ extrusion from Na+-loaded cells of V. alginolyticus 118 at pH 8.5. The results support the conclusion that Na+ extrusion from this organism at pH 8.5 occurs as a result of Na+/H+ antiport activity. Data are presented indicating the presence in V. alginolyticus 118 of an NADH oxidase which is stimulated by Na+ at pH 8.5.