Rule-based simulation of temperate bacteriophage infection: Restriction–modification as a limiter to infection in bacterial populations

An individual-based model (IbM) for bacterial adaptation and evolution, COSMIC-Rules, has been employed to simulate interactions of virtual temperate bacteriophages (phages) and their bacterial hosts. Outcomes of infection mimic those of a phage such as lambda, which can enter either the lytic or lysogenic cycle, depending on the nutritional status of the host. Infection of different hosts possessing differing restriction and modification systems is also simulated. Phages restricted upon infection of one restricting host can be adapted (by host-controlled modification of the phage genome) and subsequently propagate with full efficiency on this host. However, such ability is lost if the progeny phages are passaged through a new host with a different restriction and modification system before attempted re-infection of the original restrictive host. The simulations show that adaptation and re-adaptation to a particular host-controlled restriction and modification system result in lower efficiency and delayed lysis of bacterial cells compared with infection of non-restricting host bacteria.     

Fluorescent homogeneous immunosensors for detecting pathogenic bacteria

We developed a straightforward antibody-based assay for rapid homogeneous detection of bacteria. Our sensors utilize antibody recognizing cell-surface epitopes of the target cell. Two samples of the antibody are prepared, each labeled via nanometer size flexible linkers with short complementary oligonucleotides that are modified with fluorochromes that could participate in fluorescence resonance energy transfer (FRET). The length of the complementary oligonucleotide sequences was designed such that very little annealing occurred in the absence of the target cells. In the presence of the target cells the two labeled antibodies bind to the surface of the cell resulting in a large local concentration of the complementary oligonucleotides that are attached to the antibody. This in turn drives the annealing of the complementary oligonucleotides which brings the fluorescence probes to close proximity producing large FRET signals proportional to the amount of target cells. Long flexible linkers used to attach the oligonucleotides to the antibody enable target-induced oligonucleotide annealing even if the density of surface antigens is only modest. We used Escherichia coli 0157:H7 and Salmonella typhimurium to demonstrate that this design produced sensors exhibiting rapid response time, high specificity, and sensitivity in detecting the target bacteria.     

Biotemplates in the green synthesis of silver nanoparticles

This article recapitulates the scientific advancement towards the greener synthesis of silver nanoparticles. Applications of noble metals have increased throughout human civilization, and the uses for nano-sized particles are even more remarkable. “Green” nanoparticle synthesis has been achieved using environmentally acceptable solvent systems and eco-friendly reducing and capping agents. Numerous microorganisms and plant extracts have been applied to synthesize inorganic nanostructures either intracellularly or extracellularly. The use of nanoparticles derived from noble metals has spread to many areas including jewelery, medical fields, electronics, water treatment and sport utilities, thus improving the longevity and comfort in human life. The application of nanoparticles as delivery vehicles for bactericidal agents represents a new paradigm in the design of antibacterial therapeutics. Orientation, size and physical properties of nanoparticles influences the performance and reproducibility of a potential device, thus making the synthesis and assembly of shape- and size-controlled nanocrystals an essential component for any practical application. This need has motivated researchers to explore different synthesis protocols.     

Microbial metabolomics: past,present and future methodologies

Microbial metabolomics has received much attention in recent years mainly because it supports and complements a wide range of microbial research areas from new drug discovery efforts to metabolic engineering. Broadly, the term metabolomics refers to the comprehensive (qualitative and quantitative) analysis of the complete set of all low molecular weight metabolites present in and around growing cells at a given time during their growth or production cycle. This review focuses on the past, current and future development of various experimental protocols in the rapid developing area of metabolomics in the ongoing quest to reliably quantify microbial metabolites formed under defined physiological conditions. These developments range from rapid sample collection, instant quenching of microbial metabolic activity, extraction of the relevant intracellular metabolites as well as quantification of these metabolites using enzyme based and or modern high tech hyphenated analytical protocols, mainly chromatographic techniques coupled to mass spectrometry (LC-MSⁿ, GC-MSⁿ, CE-MSⁿ), where n indicates the number of tandem mass spectrometry, and nuclear magnetic resonance spectroscopy (NMR).     

Single-species microbial biofilm screening for industrial applications

While natural microbial biofilms often consist of multiple species, single-species biofilms are of great interest to biotechnology. The current study evaluates biofilm formation for common industrial and laboratory microorganisms. A total of 68 species of biosafety level one bacteria and yeasts from over 40 different genera and five phyla were screened by growing them in microtiter plates and estimating attached biomass by crystal violet staining. Most organisms showed biofilm formation on surfaces of polystyrene within 24 h. By changing a few simple conditions such as substratum characteristics, inoculum and nutrient availability, 66 strains (97%) demonstrated biofilm formation under at least one of the experimental conditions and over half of these strains were classified as strong biofilm formers, potentially suitable as catalysts in biofilm applications. Many non-motile bacteria were also strong biofilm formers. Biofilm morphologies were visualized for selected strains. A model organism, Zymomonas mobilis, easily established itself as a biofilm on various reactor packing materials, including stainless steel.     

Cold glycerol-saline: the promising quenching solution for accurate intracellular metabolite analysis of microbial cells

Microbial metabolomics has been seriously limited by our inability to perform a reliable separation of intra- and extracellular metabolites with efficient quenching of cell metabolism. Microbial cells are sensitive to most (if not all) quenching agents developed to date, resulting in leakage of intracellular metabolites to the extracellular medium during quenching. Therefore, as yet we are unable to obtain an accurate concentration of intracellular metabolites from microbial cell cultures. However, knowledge of the in vivo concentrations of intermediary metabolites is of fundamental importance for the characterization of microbial metabolism so as to integrate meaningful metabolomics data with other levels of functional genomics analysis. In this article, we report a novel and robust quenching method for microbial cell cultures based on cold glycerol-saline solution as the quenching agent that prevents significant leakage of intracellular metabolites and, therefore, permits more accurate measurement of intracellular metabolite concentrations in microbial cells.     

Diversity of Gut Bacteria of <Emphasis Type="Italic">Reticulitermes flavipes</Emphasis> as Examined by 16S rRNA Gene Sequencing and Amplified rDNA Restriction Analysis

The phylogenetic species richness of the bacteria in the gut of the termite Reticulitermes flavipes was examined using near full-length 16S rRNA gene sequencing and amplified rDNA restriction analysis (ARDRA). We amplified the genes by polymerase chain reaction (PCR) directly from a mixed population of termite gut bacteria and isolated them using cloning techniques. Sequence analysis of 42 clones identified a broad taxonomic range of ribotypes from six phyla within the domain Bacteria: Proteobacteria, Spirochaetes, Bacteroidetes, Firmicutes, Actinobacteria, and the recently proposed “Endomicrobia.” Analysis of the sequence data suggested the presence of a termite specific bacterial lineage within Bacteroidetes. The ARDRA data included 261 different ARDRA profiles of 512 clones analyzed. These data suggest the gut flora in R. flavipes is extremely diverse.     

Intracellular metalloporphyrin metabolism in Staphylococcus aureus

The bacterial pathogen Staphylococcus aureus is responsible for a significant amount of human morbidity and mortality, and the ability of S. aureus to cause disease is absolutely dependent on the acquisition of iron from the host. The most abundant iron source to invading staphylococci is in the form of the porphyrin heme. S. aureus is capable of acquiring nutrient iron from heme and hemoproteins via two heme-acquisition systems, the iron-regulated surface determinant system (Isd) and the heme transport system (Hts). Heme acquisition through these systems is involved in staphylococcal pathogenesis suggesting that the intracellular fate of heme plays a significant role in the infectious process. The valuable heme molecule presents a paradox to invading bacteria because although heme is an abundant source of nutrient iron, the extreme reactivity of heme makes it toxic at high concentrations. Therefore, bacteria must regulate the levels of intracellular heme to avoid toxicity. Although the molecular mechanisms responsible for staphylococcal heme acquisition are beginning to emerge, the mechanisms by which S. aureus regulate intracellular heme homeostasis are largely unknown. In this review we describe three potential fates of host-derived heme acquired by S. aureus during infection: (i) degradation for use as a nutrient iron source, (ii) incorporation into bacterial heme-binding proteins for use as an enzyme cofactor, or (iii) efflux through a dedicated ABC-type transport system. We hypothesize that the ultimate fate of exogenously acquired heme in S. aureus is dependent upon the intracellular and extracellular availability of both iron and heme.     

Plant growth-promoting rhizobacteria do not pose any deleterious effect on cowpea and detectable amounts of ethylene are produced

Summary The use of trap crops such as cowpea could reduce the effects of the root parasitic weed, Striga hermonthica and its subsequent constraints on the growth of cereals. Certain bacteria could augment the trap crop stimulatory effect. We studied the effect of three bacteria introduced to the rhizosphere of three cowpea varieties at planting. Number of days to cowpea flowering was noted and at harvest, data were collected on pod characteristics and biomass. Means of data subjected to ANOVA were compared using Tukey’s Studentized Range Test. We analysed bacterial headspace volatiles for ethylene by gas chromatography and gas chromatography–mass spectrometry. Bacterial type significantly influenced the cowpea varieties with better performance over the non-inoculated control. Average pod weight (g) with bacterial treatment was 37.97 for Enterobacter sakazakii 8MR5, 34.38 for Pseudomonas 44MS8 and 27.46 for Pseudomonas 10M3. Non-inoculated control had an average weight of 20.98 g. Bacteria promoted a significant increase in pod weight (≥30.89%), fresh biomass (≥24.22%), and improved pod number (≥20.54%) and pod wall thickness (≥7.33%) with no deleterious effect on plant health. Ethylene released by the bacteria ranged from trace concentrations in Pseudomonas sp. to 210 nmoles/10⁸ c. f. u./ml in Ent. sakazakii 8MR5.