Variations in the energy metabolism of biotechnologically relevant heterofermentative lactic acid bacteria during growth on sugars and organic acids

Heterofermentative lactic acid bacteria (LAB) such as Leuconostoc, Oenococcus, and Lactobacillus strains ferment pentoses by the phosphoketolase pathway. The extra NAD(P)H, which is produced during growth on hexoses, is transferred to acetyl-CoA, yielding ethanol. Ethanol fermentation represents the limiting step in hexose fermentation, therefore, part of the extra NAD(P)H is used to produce erythritol and glycerol. Fructose, pyruvate, citrate, and O₂ can be used in addition as external electron acceptors for NAD(P)H reoxidation. Use of the external acceptors increases the growth rate of the bacteria. The bacteria are also able to ferment organic acids like malate, pyruvate, and citrate. Malolactic fermentation generates a proton potential by substrate transport. Pyruvate fermentation sustains growth by pyruvate disproportionation involving pyruvate dehydrogenase. Citrate is fermented in the presence of an additional electron donor to acetate and lactate. Thus, heterofermentative LAB are able to use a variety of unusual fermentation reactions in addition to classical heterofermentation. Most of the reactions are significant for food biotechnology/microbiology.     

Contribution to nitrogen fixation and soil fertility by photosynthetic bacteria

Summary The nitrogen fixation ability ofRhodopseudomonas capsulatus (a member of the photosynthetic bacteria) has been investigated. This organism can fix N₂ most effectively under illuminated anaerobic conditions. However, in mixed culture in symbiotic association with heterotrophic bacteria, this microorganism using pyruvic acid excreted by the heterotrophs is capable of fixing nitrogen even under an apparent aerobic environment. It has been demonstrated that some correlation exists between the growth of photosynthetic bacteria and the reproduction of the rice plant. Compared to the mineral fertilizer, application of photosynthetic bacteria at the reproductive stage of rice plants increased the yield of grain. This was confirmed by the fact that the root system of rice is capable of absorbing amino acids and nucleic acids excreted by photosynthetic bacteria. Uracil and proline have the most influence on rice reproduction. This is also true for tomato plants. Many toxic molecules such as hydrogen sulfide, amines, etc. are found in soil. They are metabolized by photosynthetic bacteria, which contributes to the detoxication of soil. Such findings were extended to the purification of polluted waste waters from industry and domestic sources,etc. It can be concluded that photosynthetic bacteria contribute very significantly to soil fertility and improvement of the plant growth condition.     

Wild-Type Strains ofDictyostelium discoideumCan Be Transformed Using a Novel Selection Cassette Driven by the Promoter of the Ribosomal V18 Gene

Almost all methods for transformation of the social amebaDictyostelium discoideumrely on axenic growth, that is, growth in a synthetic medium, for at least part of the procedure. Axenic growth requires several mutations. Here we describe a procedure that can be used to transform wild-type strains which are able to grow only on the natural food source, bacteria. The method relies on a new selection cassette driven by the V18 promoter, a promoter that we show is substantially more active during growth on bacteria than the actin-6 promoter, which is widely used for axenic transformation. The procedure gives transformation frequencies of about 10⁻⁵with both strains Ax2 (capable of axenic growth) and NC4 (capable of growth only on bacteria). Using this vector, we have obtained NC4 strains carrying several β-galactosidase reporter cassettes. Our vector can also be used in axenic transformations.     

Growth and polyhydroxybutyrate production by Ralstonia eutropha in emulsified plant oil medium

Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by bacteria for carbon and energy storage that also have commercial potential as bioplastics. One promising class of carbon feedstocks for industrial PHA production is plant oils, due to the high carbon content of these compounds. The bacterium Ralstonia eutropha accumulates high levels of PHA and can effectively utilize plant oil. Growth experiments that include plant oil, however, are difficult to conduct in a quantitative and reproducible manner due to the heterogeneity of the two-phase medium. In order to overcome this obstacle, a new culture method was developed in which palm oil was emulsified in growth medium using the glycoprotein gum arabic as the emulsifying agent. Gum arabic did not influence R. eutropha growth and could not be used as a nutrient source by the bacteria. R. eutropha was grown in the emulsified oil medium and PHA production was measured over time. Additionally, an extraction method was developed to monitor oil consumption. The new method described in this study allows quantitative, reproducible R. eutropha experiments to be performed with plant oils. The method may also prove useful for studying growth of different bacteria on plant oils and other hydrophobic carbon sources.     

A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds

The oxazine dye Nile blue A and its fluorescent oxazone form, Nile red, were used to develop a simple and highly sensitive staining method to detect poly(3-hydroxybutyric acid) and other polyhydroxyalkanoic acids (PHAs) directly in growing bacterial colonies. In contrast to previously described methods, these dyes were directly included in the medium at concentrations of only 0.5 μg/ml, and growth of the cells occurred in the presence of the dyes. This allowed an estimation of the presence of PHAs in viable colonies at any time during the growth experiment and a powerful discrimination between PHA-negative and PHA-positive strains. The presence of Nile red or Nile blue A did not affect growth of the bacteria. This viable-colony staining method was in particular applicable to gram-negative bacteria such as Azotobacter vinelandii, Escherichia coli, Pseudomonas putida, and Ralstonia eutropha. It was less suitable for discriminating between PHA-negative and PHA-positive strains of gram-positive bacteria such as Bacillus megaterium or Rhodococcus ruber, but it could also be used to discriminate between wax-ester- and triacylglycerol-negative and -positive strains of Acinetobacter calcoaceticus or Rhodococcus opacus. The potential of this new method and its application to further investigations of PHA synthases and PHA biosynthesis pathways are discussed. Received: 12 August 1998 / Accepted: 11 November 1998     

Growth,cell division and sporulation in mycobacteria

Bacteria have the ability to adapt to different growth conditions and to survive in various environments. They have also the capacity to enter into dormant states and some bacteria form spores when exposed to stresses such as starvation and oxygen deprivation. Sporulation has been demonstrated in a number of different bacteria but Mycobacterium spp. have been considered to be non-sporulating bacteria. We recently provided evidence that Mycobacterium marinum and likely also Mycobacterium bovis bacillus Calmette–Guérin can form spores. Mycobacterial spores were detected in old cultures and our findings suggest that sporulation might be an adaptation of lifestyle for mycobacteria under stress. Here we will discuss our current understanding of growth, cell division, and sporulation in mycobacteria.     

The ins and outs of bacterial iron metabolism

Iron is a critical nutrient for the growth and survival of most bacterial species. Accordingly, much attention has been paid to the mechanisms by which host organisms sequester iron from invading bacteria and how bacteria acquire iron from their environment. However, under oxidative stress conditions such as those encountered within phagocytic cells during the host immune response, iron is released from proteins and can act as a catalyst for Fenton chemistry to produce cytotoxic reactive oxygen species. The transitory efflux of free intracellular iron may be beneficial to bacteria under such conditions. The recent discovery of putative iron efflux transporters in Salmonella enterica serovar Typhimurium is discussed in the context of cellular iron homeostasis.     

Analytical solutions for distributions of chemotactic bacteria

This paper reports general and specialized results on analytical solutions to the governing phenomenological equations for chemotactic redistribution and population growth of motile bacteria. It is shown that the number of bacteria cells per unit volume,b, is proportional to a certain prescribed function ofs, the concentration of the critical substrate chemotactic agent, for steady-state solutions through an arbitrary spatial region with a boundary that is impermeable to bacteria cell transport. Moreover, it is demonstrated that the steady-state solution forb ands is unique for a prescribed total number of bacteria cells in the spatial region and a generic Robin boundary condition ons. The latter solution can be approximated to desired accuracy in terms of the Poisson-Green's function associated with the spatial region. Also, as shown by example, closed-form exact steady-state solutions are obtainable for certain consumption rate functions and geometrically symmetric spatial regions. A solutional procedure is formulated for the initialvalue problem in cases for which significant population growth is present and bacteria cell redistribution due to motility and chemotactic flow proceeds slowly relative to the diffusion of the chemoattractant substrate. Finally, a remarkably simple exact analytical solution is reported for a stradily propagating plane-wave which features motility, chemotactic motion and bacteria population growth regulated by substrate diffusion.     

Genes specifying cytokinin biosynthesis in prokaryotes

Cytokinins are plant hormones which have long been associated with cell division and plastid differentiation. Recently, they have been found to play a central role also in the growth of plant tumors. Certain phytopathogenic bacteria, notably Agrobacterium tumefaciens and Pseudomonas syringae pv. savastanoi, can incite tumors on dicotyledonous plants and such tumors exhibit growth which is characteristic of the presence of excess auxin and cytokinin. Genes specifying cytokinin biosynthesis have now been isolated from both sets of bacteria. The genes encode prenyl transferase responsible for cytokinin biosynthesis which, upon expression in E. coli,cause the production of the active cytokinin, zeatin. Expression of these genes in association with the plant is responsible for at least part of the tumor phenotype, although the molecular mechanisms of infection by these bacteria are apparently quite dissimilar. There is extensive homology between the cytokinin biosynthetic genes from the two sets of bacteria.     

Growth patterns of yeast colonies depending on nutrient supply

Common theories of microbial growth and physiology are formulated exclusively in terms of the isolated microorganisms – especially bacteria. This is, however, an inadmissible simplification because it is obvious that the organization of microbial populations and colonies follows certain general rules. Bacterial colonies are able to generate complex interfacial growth patterns similar to those observed during diffusion-limited growth processes in non-living systems. One reason for these patterns is assumed to be the ability of many bacteria to swarm in an active manner on a substrate surface. Therefore the models of bacterial colony growth incorporate “random walkers”, which move actively in response to a gradient in the concentration of nutrients and communicate with each other by means of a chemotactic feedback. A selected number of yeasts were tested with regard to their colony growth patterns depending on the medium parameters such as nutrient concentration. Growth patterns similar to those which were described in literature for bacteria were also found in these experiments. It concerns in particular growth types like compact growth, fractal growth and dense-branching growth. This result allows a hypothesis to be formulated, that – especially in the case of fractal growth patterns – wandering of cells on a substrate surface may be induced by uncontrolled “swimming” on a thin water film caused by the metabolic activity (e.g. respiration) of the cells on the surface of the agar. Furthermore it was found that an interplay between changes in the individual morphology of yeast cells and the morphology transitions takes place. Such growth patterns are known for Candida sp. which are able to form pseudomycel and blastospores.     

Roles of Environmental Heme, and Menaquinone, in Streptococcus Agalactiae

Most bacteria require iron for growth. However, as it may not be directly available under aerobic conditions, bacteria may use iron-sequestering molecules, such as bacterially encoded siderophores, or heme, which is the major iron source in the animal host. Bacteria may also assimilate heme for purposes other than as an iron source. Once internalised, heme can activate, for example, a heme-dependent catalase and a cytochrome oxidase. In bacterial pathogen Streptococcus agalactiae, heme, in association with exogenous menaquinone, activates a respiratory chain. Respiration has radical effects on carbon metabolism. GBS respiration-grown cells display improved survival in an aerobic environment and greater virulence in a murine septicemia model. GBS might benefit from its ecological niches to capture heme and menaquinone, i.e., from other bacteria when it colonizes host mucosa, or from blood-containing organs during septicemia.     

Saprospira species—Natural predators

SomeSaprospira spp. from fresh waters have been shown to be predatory on other aquatic bacteria, particularly gliding bacteria. Some can be grown as well axenically but others so far have been cultured only on the host bacteria for which they show a degree of specificity. A marine strain resemblingSaprospira grandis, although capable of growth axenically, is also a predator on a marineCytophaga sp.S. grandis ATCC 23119 failed to grow on the base media or media overgrown with host cells of otherSaprospira spp.     

Monitoring of biofilms grown on differentially structured metallic surfaces using confocal laser scanning microscopy

Imaging of biofilms on opaque surfaces is a challenge presented to researchers especially considering pathogenic bacteria, as those typically grow on living tissue, such as mucosa and bone. However, they can also grow on surfaces used in industrial applications such as food production, acting as a hindrance to the process. Thus, it is important to understand bacteria better in the environment they actually have relevance in. Stainless steel and titanium substrata were line structured and dotted surface topographies for titanium substrata were prepared to analyze their effects on biofilm formation of a constitutively green fluorescent protein (GFP)‐expressing Escherichia coli strain. The strain was batch cultivated in a custom built flow cell initially for 18 h, followed by continuous cultivation for 6 h. Confocal laser scanning microscopy (CLSM) was used to determine the biofilm topography. Biofilm growth of E. coli GFPmut2 was not affected by the type of metal substrate used; rather, attachment and growth were influenced by variable shapes of the microstructured titanium surfaces. In this work, biofilm cultivation in flow cells was coupled with the most widely used biofilm analytical technique (CLSM) to study the time course of growth of a GFP‐expressing biofilm on metallic surfaces without intermittent sampling or disturbing the natural development of the biofilm.     

Isolation and characterization of autotrophic, hydrogen-utilizing, perchlorate-reducing bacteria

Recent studies have shown that perchlorate (ClO₄ ^–) can be degraded by some pure-culture and mixed-culture bacteria with the addition of hydrogen. This paper describes the isolation of two hydrogen-utilizing perchlorate-degrading bacteria capable of using inorganic carbon for growth. These autotrophic bacteria are within the genus Dechloromonas and are the first Dechloromonas species that are microaerophilic and incapable of growth at atmospheric oxygen concentrations. Dechloromonas sp. JDS5 and Dechloromonas sp. JDS6 are the first perchlorate-degrading autotrophs isolated from a perchlorate-contaminated site. Measured hydrogen thresholds were higher than for other environmentally significant, hydrogen-utilizing, anaerobic bacteria (e.g., halorespirers). The chlorite dismutase activity of these bacteria was greater for autotrophically grown cells than for cells grown heterotrophically on lactate. These bacteria used fumarate as an alternate electron acceptor, which is the first report of growth on an organic electron acceptor by perchlorate-reducing bacteria.     

Growth promotion of <Emphasis Type="Italic">Xanthium italicum</Emphasis> by application of rhizobacterial isolates of <Emphasis Type="Italic">Bacillus aryabhattai</Emphasis> in microcosm soil

This study was conducted using rhizobacteria, which are able to exert beneficial effects upon plant growth in the infertile soil collected from barren lakeside areas. Four strains of plant growth promoting bacteria were isolated from the rhizosphere of a common wild plant, Erigeron canadensis. Isolated strains LS9, LS11, LS12, and LS15 were identified as Bacillus aryabhattai by 16S rDNA sequence analysis. B. aryabhattai LS9, LS11, LS12, and LS15 could solubilize 577.9, 676.8, 623.6, and 581.3 mg/L of 0.5% insoluble calcium phosphate within 2 days of incubation. Production of indole acetic acid, a typical growth promoting phytohormone auxin, by strain LS15 was 471.3 mg/L in 2 days with the addition of auxin precursor L-tryptophan. All the strains also produced other phytohormones such as indole butyric acid, gibberellins, and abscisic acid, and strain LS15 showed the highest production rate of gibberellin (GA₃), 119.0 μg/mg protein. Isolated bacteria were used in a microcosm test for growth of wild plant Xanthium italicum, which can be utilized as a pioneer plant in barren lands. Seed germination was facilitated, and the lengths of roots, and shoots and the dry weights of germinated seedlings after 16 days were higher than those of the uninoculated control plants. Root lengths of seedlings of X. italicum increased by 121.1% in LS11-treated samples after 16 days. This plant growth-promoting capability of B. aryabhattai strains may be utilized as an environmentally friendly means of revegetating barren lands, especially sensitive areas such as lakeside lands.     

Quantification of factors affecting the probability of development of pathogenic bacteria,in particularClostridium botulinum,in foods

Summary The success of a preservation method with respect toClostridium botulinum can be measured by its effect on the probability that a single spore would result in growth and formation of toxin. In canned, low-acid foods, the minimum heat-process is designed to reduce the probability of survival of a single, heat-resistant spore ofC. botulinum by a factor of 10¹². In some foods, safety depends on the combination of inactivation and inhibition ofC. botulinum. The degree of protection (Pr) can be expressed asPr=Ds+In, whereDs is the decimal destruction of spores ofC. botulinum andIn is the decimal inhibition. A similar approach can be used in the case of other bacteria.Pr=log 1/P, whereP=the probability that an individual spore or bacterium will survive and result in growth.P can be estimated as the number of spores or bacteria that survive and initiate growth in a culture medium or food divided by the number of viable spores or bacteria inoculated into the medium or food. The effects of combinations of preservative factors can be measured by their effect onP at a stated temperature for a stated time. In experiments to determine the effects of preservative factors on an anaerobic bacterium such asC. botulinum it is essential that oxygen should be eliminated, unless it is controlled as an inhibitory factor. Thus experiments in culture media should be done under strictly anaerobic conditions at a known, low redox potential. The results of experiments to determine the effects of preservative factors onP after a series of incubation times can be modeled by methods similar to those used to model the effect of preservative factors on rate of growth and on lag period. Experiments to determine the effect of preservative factors on the probability of growth from a single spore or bacterium ofC. botulinum are discussed. A few reports of similar experiments with other bacteria have been published and are described. This approach has the advantage that it takes account of the inoculum level of bacteria.Mention of brand of firm names does not constitute an endorsement by the US Department of Agriculture over others of a similar nature not mentioned.     

In vitro analyses are not reliable predictors of the plant growth promotion capability of bacteria; a Pseudomonas fluorescens strain that promotes the growth and yield of wheat

Aims: In this study, we set out to identify bacteria that can be used to promote the growth of cereals, while concurrently investigating the merits of using a range of such tests to preselect bacteria for glasshouse studies. Methods and Results: A panel of 15 strains isolated from the rhizosphere and phyllosphere of cereals was tested for the ability to improve the germination of wheat seeds and for production of a range of factors associated with plant growth promotion. In parallel, all bacteria were tested for their ability to improve biomass and grain yield when applied as a soil amendment in glasshouse trials. Conclusions: There was no significant correlation between growth promotion potential in the glasshouse and the results of either the phenotypic or the germination tests. Glasshouse tests identified that only one strain, Pseudomonas fluorescens strain MKB37, gave a significant increase in head weight and grain yield. Significance and Impact of the Study: While this study has identified a candidate for further field tests, it has also highlighted the fact that the modes of action for plant growth‐promoting bacteria (PGPB) are still not fully understood, and that there is no efficient and effective screening method for identifying PGPB by laboratory tests.     

Growth of Leptospirillum ferriphilum in sulfur medium in co-culture with Acidithiobacillus caldus

Leptospirillum ferriphilum and Acidithiobacillus caldus are both thermotolerant acidophilic bacteria that frequently co-exist in natural and man-made environments, such as biomining sites. Both are aerobic chemolithotrophs; L. ferriphilum is known only to use ferrous iron as electron donor, while A. caldus can use zero-valent and reduced sulfur, and also hydrogen, as electron donors. It has recently been demonstrated that A. caldus reduces ferric iron to ferrous when grown aerobically on sulfur. Experiments were carried out which demonstrated that this allowed L. ferriphilum to be sustained for protracted periods in media containing very little soluble iron, implying that dynamic cycling of iron occurred in aerobic mixed cultures of these two bacteria. In contrast, numbers of viable L. ferriphilum rapidly declined in mixed cultures that did not contain sulfur. Data also indicated that growth of A. caldus was partially inhibited in the presence of L. ferriphilum. This was shown to be due to greater sensitivity of the sulfur-oxidizer to ferric than to ferrous iron, and to highly positive redox potentials, which are characteristic of cultures containing Leptospirillum spp. The implications of these results in the microbial ecology of extremely acidic environments and in commercial bioprocessing applications are discussed.

     

Species differentiation of insects and other multicellular organisms using matrix-assisted laser desorption/ionization time of flight mass spectrometry protein profiling

Abstract. Protein profiling by ‘whole cell MALDI’ is a well developed tool for species identification in bacteria but this is the first report that this inexpensive and simple technique can also be used for the identification of small multicellular organisms such as insects. A proof-of-concept experiment demonstrates that the method can distinguish between sibling species from the Drosophila melanogaster subgroup. This suggests that the technique may be used as a rapid screen for cryptic species and to identify known species.     

A simple and rapid method for the elimination of R plasmids from enteric bacteria

A rapid, simple, and effective method for the curing of a wide range ofEscherichia coli antibiotic resistance plasmids is described. Treatment with acridine orange followed by growth in sublethal concentration of antibiotics and penicillin selection under such bacteriostatic conditions resulted in a curing efficiency of more than 98% in all cases tested. The method is equally applicable, with modifications, to other enteric bacteria such asKlebsiella pneumoniae. It is also equally applicable to nutritional markers for which toxic analogues exist, and to elimination of recombinant bacteriophages containing antibiotic resistance transposons.