Physiology of biofilms of thermophilic bacilli—potential consequences for cleaning

Thermophilic Bacillus species readily attached and grew on stainless steel surfaces, forming mature biofilms of >10^6.0 cells/cm² in 6 h on a surface inoculated with the bacteria. Clean stainless steel exposed only to pasteurized skim milk at 55 °C developed a mature biofilm of >10^6.0 cells/cm² within 18 h. When bacilli were inoculated onto the steel coupons, 18-h biofilms were 30 m thick. Biofilm growth followed a repeatable pattern, with a reduction in the numbers of bacteria on the surface occurring after 30 h, followed by a recovery. This reduction in numbers was associated with the production of a substance that inhibited the growth of the bacteria. Variations in the environment, including pH and molarity, affected the viability of the cells. Chemicals that attack the polysaccharide matrix of the biofilm were particularly effective in killing and removing cells from the biofilm, demonstrating the importance of polysaccharides in the persistence of these biofilms. Treatment of either the biofilm or a clean stainless steel surface with lysozyme killed biofilm cells and prevented the attachment of any bacteria exposed to the surface. This suggests that lysozyme may have potential as an alternative control method for biofilms of these bacteria.     

Datura innoxia— a potential commercial source of scopolamine

The drug scopolamine is important, among other uses, as a pre-anesthetic in surgery and childbirth, in ophthalmology and in prevention of motion sickness. Datura Metel of Asia and Africa has been regarded as the best source of it, but a pharmacognostic study, reported here, indicates that D. innoxia of Latin America is a possible industrial source.     

Fungi,a neglected component of acidophilic biofilms: do they have a potential for biotechnology?

Extremophiles - Fungi from extreme environments, including acidophilic ones, belong to biotechnologically most attractive organisms. They can serve as a source of enzymes and metabolites with...     

Are bacteria an important food source for rotifers in eutrophic lakes?

In situ grazing measurements using fluorescent particles of0.5, 2.4 and 6.3 µm diameter in eutrophic Lake Loosdrecht(The Netherlands) showed that Anuraeopsis fissa, a small rotifer,filtered the smallest, bacteria-sized particles as efficientlyor more efficiently than the larger particles. In contrast,three other rotifer species (Brachionus angularis, Filinia longisetaand Pompholyx sulcata) filtered the bacteria-sized particlesless efficiently than the larger particles. Both Keratella cochlearisand Conochilus unicornis only ingested the bacteria-sized particles.Anuraeopsis fissa had a higher uptake of fluorescent bacteria-sizedparticles than K.cochlearis, both in 1 µm filtrate oflake water and in lake water. Within both species, uptake didnot differ between juveniles and adults. When cultured on threedifferent size fractions of lake water (1, 3 and 15 µmfiltrate) in July, all rotifer species declined in numbers onthe 1 and 3 µm filtrates, while A.fissa and B.angularisincreased in numbers on the 15 µm filtrate. The high abundanceof small bacteria in the lake water could not support rotiferpopulations. It is concluded that bacteria are not a suitablefood source of high quality for A.fissa because its populationdoes not grow even though the bacterial concentration was higherthan its estimated threshold food concentration. In August,when individually cultured, the mortality was high for all species,but especially for F.longiseta. The lifespan of K.cochleariswas reduced in the 1 and 3 µm filtrates of lake water,compared with in the 15 µm filtrate. The lifespan of A.fissawas similar in all filtrates, but reproduction was reduced inthe 1 and 3 µm filtrates, as in Keratella. On the 15 µmfiltrate, their ages at first reproduction and growth ratesdid not differ. Individuals of A.fissa older than 4 days showeda higher survival in the 15 µm filtrate than in the othertwo filtrates, as did K.cochlearis throughout its life. Hence,bacteria seem to be a more important food source for youngerindividuals of A.fissa than of K.cochlearis.     

RhoGTPases — NODes for effector-triggered immunity in animals

A recent study published in Nature by Keestra and colleagues addresses how the immune system detects the pathogenic potential of microbes and provides evidence that one strategy involves NOD1, which monitors the activation state of the RhoGTPases that are targeted by virulence effectors produced by pathogenic microbes. Interestingly, their findings reveal striking similarities with previous observations made in flies and plants, establishing the evolutionary conservation of this detection system in the innate immune arsenal in many taxa.The discovery that Drosophila Toll, and the homologous Toll-like receptors (TLRs) in animals, are pattern recognition receptors (PRRs) that act as cellular sensors of microbes has attracted considerable attention during the last two decades¹. The PRR system is based on the detection of conserved molecular motifs, microbe-associated molecular patterns (MAMPS), that are shared by most microbes, virulent or not. Given the conserved nature of MAMPS, this model does not explain how the host discriminates between harmful pathogens and beneficial commensal microbes. An elegant hypothesis is that, in addition to the PRR system, the host is able to monitor the pathogen-induced disruption of cellular homeostasis. This type of immune surveillance system has been demonstrated in plants and termed “effector-triggered immunity” (ETI)². Recently, the concept of ETI has been extended to metazoans and proof of its importance as an innate immune mechanism has now been provided in Drosophila melanogaster, Caenorabditis elegans and mammals³.The ETI model is of particular relevance when considering that most major pathogenic bacteria have evolved many protein effectors commonly referred to as virulence factors. These effectors are either directly injected into host cells by cell-bound bacteria, or are secreted toxins endowed with the ability to bind to and translocate into the host cell cytosol. Once within the host cell, these bacterial effectors perturb cellular homeostasis by modifying the activity of critical regulators. Common amongst the arsenal of numerous pathogenic bacteria are virulence factors that target the small RhoGTPases of the host⁴. This predilection for targeting RhoGTPases is probably because it allows bacteria to hijack the many cellular functions that contribute to immunity including phagocytosis, apoptosis, as well as production of reactive oxygen species (ROS) and inflammatory mediators⁵. In this regard, the RhoGTPases represent a common target and vulnerability in the host cell, which explains why their aberrant activity can often indicate pathogen invasion.Providing the foundation for the work of Keestra et al.⁶, previous studies have shown that effectors that activate RhoGTPase can induce unusual immune responses in the host. Both Salmonella typhimurium and Shigella spp. are enteric pathogens that invade host cells using a type III secretion system that is able to inject effectors in the cytosol of host cells. Earlier work by the group of Jorge Galan showed that Salmonella effectors could activate epithelial cells through a PRR-independent mechanism that was dependent on the GEF activity of certain effectors and involving target GTPases in the host⁷. Similarly, the Shigella effector, GEF-H1 was shown to augment NF-κB-dependent immune responses in a NOD1-dependent manner also after modifying RhoGTPases⁸. Finally, the activation of RacGTPase by the CNF1 toxin of uropathogenic Escherichia coli also triggers an immune response⁹. In this case the response is via an innate immune signaling pathway conserved in Drosophila and mammals involving IMD in flies and the related Rip proteins, RIP1 and RIP2, in mammalian cells⁹. Moreover, this response can be beneficial for the host and help clear the bacteria as shown in an in vivo fly model. Now, using a mouse model of Salmonella typhimurium infection, Keestra et al.⁶ further define the mechanism of detection of effectors that target RhoGTPase in vertebrates. They show that mammals detect the activity of the injected effector SopE once it is active in the host cytosol. Using cell-based assays, they demonstrate that this detection mechanism is through NOD1, a NOD-Like Receptor (NLR) protein, in a molecular complex containing HSP90. Together this complex detects the activation of the RhoGTPases Rac1 and Cdc42 and transduces a danger signal though RIP2 kinase. Furthermore, using NOD1-deficient mice, they show that SopE-triggered inflammation is markedly reduced.Together this emerging body of data support the idea that the activity of GTPases is monitored by NLR and related pathways, and used as a cue to augment ongoing immune responses during pathogen invasion. Interestingly, Kawano and colleagues have shown that resistance to the rice blast fungus also involves activation of Rac (OsRac1) downstream of Pit, a plant nucleotide-binding site-leucine-rich repeat (NBS-LRR) receptor¹⁰. OsRac1 contributes to NBS-LRR-mediated production of ROS and induction of a hypersensitive response for the purpose of destroying infected tissue and preventing dissemination. Although the link between RhoGTPases and NLRs in mammals and NBS-LRR in plants is likely a consequence of convergent evolution, these data highlight some striking similarities between the two systems (Figure 1). Together these papers suggest that we should now consider the NOD proteins not only as intracellular PRR but also akin to plant NBS-LRRs that are able to sense the direct and indirect perturbations of host cell homeostasis. Moreover, these data place RhoGTPases as central players in the molecular cascades of ETI in many species, explaining why monitoring the activation state of RhoGTPases as a surrogate for the presence of virulent pathogens is an evolutionarily conserved strategy. Our current challenge will now be to determine how PRR- and effector-triggered immunity collaborate to confer optimal protection during infections with virulent pathogens.Open in a separate windowFigure 1RhoGTPases are components of effector-triggered immune responses in different species. The role of the RhoGTPases OsRac1 in plants (left), Rac2 in flies and mammals (middle), and Rac1 and CDC42 in mammals (right) in ETI responses. Activation of Rac2 by CNF1 (middle), and Rac1 and CDC42 by SopE engages RIP kinase-dependent signaling pathways. In plants OsRac1 is activated downstream of the NBS-LRR, Pit, and the response to SopE (right panel) requires the NLR, NOD1. In contrast, flies lack NLRs and the ETI response occurs independently of NLRs but does require the RhoGTPase, Rac2. Middle and right panels — inactive GTPase bound to GDP is shown in grey (black square) and the active GTPase bound to GTP in blue (blue circle).     

Tricin—a potential multifunctional nutraceutical

This review throws light on the natural occurrence and distribution of tricin (5,7,4′-trihydroxy-3′,5′-dimethoxyflavone) and its conjugated forms, as more common natural plant constituents than previously known. It examines the current literature dealing with its biosynthesis, regulation, biological significance, pharmacological effects, and potential role as a chemopreventive and anticancer agent. Because of its common occurrence in cereal grain plants and the wide spectrum of its health promoting effects, a metabolic engineering strategy is proposed to produce tricin in sufficient amounts for further experimentation, and increase its accumulation in wheat grain endosperm as a nutraceutical.     

Is there a role for quorum sensing signals in bacterial biofilms?

Bacteria form multicellular biofilm communities on most surfaces. Genetic analysis of biofilm formation has led to the proposal that extracellular signals and quorum-sensing regulatory systems are essential for differentiated biofilms. Although such a model fits the concept of density-driven cell-cell communication and appear to describe biofilm development in several bacterial species and conditions, biofilm formation is multifactorial and complex. Hydrodynamics, nutrient load and intracellular carbon flux have major impacts, presumably by altering the expression of cellular traits essential for bacterial adaptation during the different stages of biofilm formation. Hence, differentiated biofilms may also be the net result of many independent interactions, rather than being determined by a particular global quorum sensing system.     

Plants and animals: a different taste for microbes?

Plants and animals can recognize potential pathogens by detecting pathogen-associated molecular patterns (PAMPs). Significant advances over the past few years have begun to unveil the molecular basis of PAMP perception by pattern recognition receptors (PRRs). Although these discoveries highlight common recognition strategies among higher eukaryotes, they also show differences with respect to the nature of the receptors involved and the exact molecular patterns recognized. This suggests a convergent evolution of microbe sensing by the innate immune systems of these various organisms.     

An ignored and potential source of taste and odor (T&O) issues—biofilms in drinking water distribution system (DWDS)

It is important for water utilities to provide esthetically acceptable drinking water to the public, because our consumers always initially judge the quality of the tap water by its color, taste, and odor (T&O). Microorganisms in drinking water contribute largely to T&O production and drinking water distribution systems (DWDS) are known to harbor biofilms and microorganisms in bulk water, even in the presence of a disinfectant. These microbes include T&O-causing bacteria, fungi, and algae, which may lead to unwanted effects on the organoleptic quality of distributed water. Importantly, the understanding of types of these microbes and their T&O compound-producing mechanisms is needed to prevent T&O formation during drinking water distribution. Additionally, new disinfection strategies and operation methods of DWDS are also needed for better control of T&O problems in drinking water. This review covers: (1) the microbial species which can produce T&O compounds in DWDS; (2) typical T&O compounds in DWDS and their formation mechanisms by microorganisms; (3) several common factors in DWDS which can influence the growth and T&O generation of microbes; and (4) several strategies to control biofilm and T&O compound formation in DWDS. At the end of this review, recommendations were given based on the conclusion of this review.

     

Everolimus and zoledronic acid——a potential synergistic treatment for lung adenocarcinoma bone metastasis

Non-small-cell lung cancer （NSCLC） frequently metasta- sizes to bone. It is known that zoledronic acid is cytostatic to tumors, and everolimus, the inhibitor for mammalian target of the rapamycin, could inhibit many types of cancer. Herein, we evaluated the effect of zoledronic acid alone and in combination with everolimus on treating lung adenocarcinoma bone metastasis in vitro and in vivo. Mice treated with zoledronic acid in combination with everoli- mus had more apoptotic lung cancer cells and more cells were arrested in the G1/G0 phase. The phosphorylation of p70S6K was inhibited in the combination treatment group. Lung cancer cell invasion was also significantly inhibited in the group with combination treatment in vitro. Bone nuclear scans revealed more metastatic lesions in controls compared with those in the combination treatment group. Bone scans and radiographic images indicated that com- bination therapy significantly reduced bone metastasis. The moderate survival rate suggested that the drug com- bination was synergistic, which can delay NSCLC bone metastasis and prolong survival in vivo.     

Agrobacterium rhizogenes—transformed plant roots as a source of grapevine viruses for purification

Agrobacterium rhizogenes-mediated transformation was applied toVitis spp. andNicotiana spp. infected by different grapevine phloem-limited viruses (grapevine fleck virus, grapevine virus A, grapevine virus B) to obtain root cultures for virus purification. All plant species were successfully transformed, and several clones were established in liquid culture. Transformed grapevine roots contained as much virus as non transformed roots and more than leaves, as assessed by ELISA and thin sectioning. Likewise, transformed roots ofNicotiana benthamiana Domin. contained in average more GVA than leaves, especially those at the base and the top of the plant, whereas withNicotiana occidentalis wheel., GVB was apparently less concentrated than in leaves.Nicotiana root grew faster than those ofVitis. All viruses multiplied and persisted in root cultures, which were successfully used for purification. Virus yields were the same (GFkV and GVB) or higher (GVB) than those reported in the literature. Grapevine roots may prove useful for culturing and purifying other non-mechanically transmissible grapevine viruses.     

Streaming potential—a general affinity sensor

Electrokinetic phenomena, such as streaming potential or streaming current, have so far been used for studies of unspecific adsorption of ionic compounds on various materials. This paper shows that streaming potential measurements can also be used for studies of biospecific interactions.Several different interactions were studied, e.g. lectin-carbohydrate, IgG-Protein A. Regardless of the type of interaction an increasing change in streaming potential was obtained with increasing concentration of the interacting molecule. The authors also observed a correlation between streaming potential and affinity constants for carbohydrate-induced desorption of Concanavalin A from partially hydrolyzed Sephadex G50. In conclusion, it is shown that streaming potential measurements can be used to study molecular interactions, and to determine concentrations and relative binding constants of interacting molecules.     

Alcohol oxidase from the yeast Pichia pastoris—a potential catalyst for organic synthesis

Alcohol oxidase (AO) from the methylotropic yeast Pichia pastoris was isolated and investigated. Wide substrate specificity is characteristic for this enzyme. Unbranched primary alcohols are effectively oxidized by AO to aldehydes, including propargyl alcohol, 2-chloroethanol, 2-cyanoethanol, leading to important synthetic intermediates. AO was immobilized by covalent linking to macroporous cellulose activated by glutaraldehyde, yield of immobilization 80%. Presence of two izoenzymes of AO was suggested from the pH activity dependence.