A high throughput Nile red fluorescence method for rapid quantification of intracellular bacterial polyhydroxyalkanoates

A rapid quantitative measurement of accumulated polyhydroxyalkanoate (PHA) is essential for rapid monitoring of PHA production by microorganisms. In the present study, a 96-well microplate was used as a high throughput means to measure the fluorescence intensity of the Nile red stained cells containing PHA. The linear correlation obtained between intracellular PHA concentration and the fluorescence intensity represents the potential of the Nile red method employment to determine PHA concentration. The optimal ranges of excitation and emission wavelengths were determined using bacterial cells containing different types of PHAs, of different co-monomers and compositions. Interestingly, in spite of different co-monomers compositions in each PHA, all tested PHAs fluoresced maximally at excitation wavelength between 520 and 550 nm, and emission wavelength between 590 and 630 nm. The developed staining method also had successfully demonstrated a good correlation between the amount of accumulated PHA based on the fluorescence intensity measurements and that from chromatographic analysis to evaluate poly(3-hydroxybutyrate) [P(3HB)], poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)], poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)], using the same calibration curve, despite of different co-monomers that the PHA consist. Strongly supported by these experimental results, it can therefore be concluded that the developed staining method can be efficiently applied for rapid monitoring of PHA production.     

Fluorescence-based bacterial overlay method for simultaneous in situ quantification of surface-attached bacteria

For quantification of bacterial adherence to biomaterial surfaces or to other surfaces prone to biofouling, there is a need for methods that allow a comparative analysis of small material specimens. A new method for quantification of surface-attached biotinylated bacteria was established by in situ detection with fluorescence-labeled avidin-D. This method was evaluated utilizing a silicon wafer model system to monitor the influences of surface wettability and roughness on bacterial adhesion. Furthermore, the effects of protein preadsorption from serum, saliva, human serum albumin, and fibronectin were investigated. Streptococcus gordonii, Streptococcus mitis, and Staphylococcus aureus were chosen as model organisms because of their differing adhesion properties and their clinical relevance. To verify the results obtained by this new technique, scanning electron microscopy and agar replica plating were employed. Oxidized and poly(ethylene glycol)-modified silicon wafers were found to be more resistant to bacterial adhesion than wafers coated with hydrocarbon and fluorocarbon moieties. Roughening of the chemically modified surfaces resulted in an overall increase in bacterial attachment. Preadsorption of proteins affected bacterial adherence but did not fully abolish the influence of the original surface chemistry. However, in certain instances, mostly with saliva or serum, masking of the underlying surface chemistry became evident. The new bacterial overlay method allowed a reliable quantification of surface-attached bacteria and could hence be employed for measuring bacterial adherence on material specimens in a variety of applications.     

Protocol for rapid fluorescence in situ hybridization of bacteria in cryosections of microarthropods

A protocol was developed to detect bacteria inhabiting microarthropods by means of small-subunit rRNA-targeted fluorescence in situ hybridization and microscopy. The protocol is based on cryosections of whole specimens. In contrast to more commonly applied paraffin-embedding techniques, the protocol is quicker and reduces the number of manipulations which might damage the microscopic material. The method allowed the study of the bacterial colonization of Folsomia candida (Collembola) and the detection of bacteria in both the gut and tissue.     

New protocol for the rapid quantification of exopolysaccharides in continuous culture systems of acidophilic bioleaching bacteria

In this study, we investigate exopolysaccharide production by a bacterial consortium during the bioleaching of a cobaltiferrous pyrite. Whereas comparable studies have looked at exopolysaccharide production in batch systems, this study focuses on a continuous system comprising a series of four stirred bioreactors and reveals the difficulties in quantifying biomolecules in complex media such as bioleached samples. We also adapted the phenol/sulphuric acid method to take into account iron interference, thus establishing a new protocol for sugar quantification in bioleached samples characterised by low pH (1.4) and high iron concentration (2 g l⁻¹). This allows sugar analysis without any prior sample preparation step; only a small amount of sample is needed (0.5 ml) and sample preparation is limited to a single filtration step. We found that free exopolysaccharides represented more than 80% of the total sugars in the bioreactors, probably because stirring creates abrasive conditions and detaches sugars bound to pyrite or bacteria and that they were produced mainly in the first two reactors where bioleaching activity was greatest. However, we could not establish any direct link between the measured exopolysaccharide concentration and bioleaching activity. Exopolysaccharides could have another role (protection against stress) in addition to that in bacterial attachment.     

Monitoring the identity of bacteria using their intrinsic fluorescence

Three different fluorescence spectra were recorded following excitation at 250 nm (aromatic amino acids+nucleic acids), 270 nm (tryptophan residues) and 316 nm (NADH) for 25 strains of bacteria in dilute suspensions. Evaluation of the spectra using principal component analysis and hierarchical clustering showed a good reproducibility from culture to culture and a good discrimination of the bacteria. Applying the method of Mahalanobis distances to the spectra of lactobacilli species recorded following excitation at 250 nm, a good classification was observed for 100% and 81% of calibration and validation groups, respectively. The developed method allows the discrimination and identification of the investigated bacteria at the genus, species and strain levels.     

A rapid flow cytometry assay for the relative quantification of protein encapsulation into bacterial microcompartments

Bacterial microcompartments (MCPs) are protein‐based organelles that have been suggested as scaffolds for creating in vivo nanobioreactors. One of the key steps towards engineering MCPs is to understand and maximize the encapsulation of enzymes into the lumen of the MCP. However, there are currently no high‐throughput methods for investigating protein encapsulation. Here, we describe the development of a rapid in vivo assay for quantifying the relative amount of protein encapsulated within MCPs based on fluorescence. In this assay, we fuse a degradation peptide to a MCP‐targeted fluorescence reporter and use flow cytometry to measure overall fluorescence from the encapsulated, protected reporter protein. Using this assay, we characterized various MCP‐targeting signal sequence mutants for their ability to encapsulate proteins and identified mutants that encapsulate a greater amount of protein than the wild type signal sequence. This assay is a powerful tool for reporting protein encapsulation and is an important step towards encapsulating metabolic enzymes into MCPs for synthetic biochemical pathways.     

A rapid method for detecting bacterial polyhydroxyalkanoates in intact cells by Fourier transform infrared spectroscopy

Polyhydroxyalkanoates (PHA) are synthesized by many bacteria as inclusion bodies, and their biodegradability and structural diversity have been studied with a view to their potential application as biodegradable materials. In this paper, Fourier-transform infrared spectroscopy (FT-IR) was used to carry out rapid qualitative analysis of PHA in intact bacterial cells. The FT-IR spectra of pure PHA containing short-chain-length monomers, such as hydroxybutyrate (HB), medium-chain-length hydroxyalkanoate (mclHA) monomers including hydroxyoctanoate (HO) and hydroxydecanoate (HD), or both HB and mclHA monomers, showed their strong characteristic band at 1728 cm⁻¹, 1740 cm⁻¹ or 1732 cm⁻¹ respectively. Other accompanying bands near 1280 cm⁻¹ and 1165 cm⁻¹ helped identify the types of PHA. The intensity of the methylene band near 2925 cm⁻¹ provided additional information for PHA characterization. In comparison, bacterial cells accumulating the above PHA also showed strong marker bands at 1732 cm⁻¹, 1744 cm⁻¹ or 1739 cm⁻¹, corresponding to intracellular PHB, mclPHA and P(HB + mclHA) respectively. The accompanying bands visible in pure PHA were also observable in the intact cells. The FT-IR results were further confirmed by gas chromatography analysis. Received: 14 October 1998 / Received revision: 29 December 1998 / Accepted: 30 December 1998     

Simple and sensitive bacterial quantification by a flow-based kinetic exclusion fluorescence immunoassay

A flow-based immunoassay system utilizing secondary-antibody coated microbeads and Cy5-secondary antibody for signal production was successfully developed to quantitate target bacteria with a kinetic exclusion assay (KinExA 3000 Instrument). It directly measured the concentration of unliganded antibody separated from the equilibrated mixture of antibody and bacteria through a 0.2 microm polyethersulfone membrane, enabling it to quantify the concentration of bacteria. The novel method demonstrated the qualities of rapidness, sensitivity, high accuracy and reproducibility, and ease to perform. Detection of Pseudomonas aeruginosa and Staphylococcus aureus was accomplished with low detection limits of 4.10 x 10(6) and 5.20 x l0(4)cells/mL, respectively, with an assay time of less than 15 min. The working ranges for quantification were 4.10 x l0(6) to 1.64 x l0(10)cells/mL for P. aeruginosa, and 5.20 x l0(4) to 1.04 x l0(9)cells/mL for S. aureus. It yielded an assay with at least 10-fold greater sensitivity than ELISA and could correctly assess the concentration of predominant bacterium spiked in the mixture of P. aeruginosa and S. aureus. With this reliable platform, the average amount of antibody bound by one cell in the maximum capability could be further provided: (1.6-2.5) x l0(5) antibodies for one P. aeruginosa cell and (2.2-2.7) x l0(8) antibodies for one S. aureus cell. The KinExA system is flexible to determine different kinds of bacteria conveniently by using anti-mouse IgG as the same immobilizing agent. However, a higher specificity of the antibodies to the target bacteria will be required for the use of this system with higher detection sensitivity.     

A molecular phylogeny of enteric bacteria and implications for a bacterial species concept

A molecular phylogeny for seven taxa of enteric bacteria (Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae, and Serratia plymuthica) was made from multiple isolates per taxa taken from a collection of environmental enteric bacteria. Sequences from five housekeeping genes (gapA, groEL, gyrA, ompA, and pgi) and the 16S rRNA gene were used to infer individual gene trees and were concatenated to infer a composite molecular phylogeny for the species. The isolates from each taxa formed tight species clusters in the individual gene trees, suggesting the existence of 'genotypic' clusters that correspond to traditional species designations. These sequence data and the resulting gene trees and consensus tree provide the first data set with which to assess the utility of the recently proposed core genome hypothesis (CGH). The CGH provides a genetically based approach to applying the biological species concept to bacteria.     

Structure of bacterial L forms and their parent bacteria

Weibull, Claes (Rocky Mountain Laboratory, Hamilton, Mont.). Structure of bacterial L forms and their parent bacteria. J. Bacteriol. 90:1467-1480. 1965.-Light and electron microscopic studies were done on normal cells and L forms of Proteus mirabilis, Staphylococcus aureus, and Corynebacterium sp. grown in liquid media. Under the prevailing growth conditions, the L forms studied were morphologically indistinguishable from one another. They appeared as approximately spherical elements occurring singly or more often connected with each other by thinner portions of cell material. In sections of large L forms, the following structures were seen: a peripheral, triple-layered ("unit") membrane, a granular cytoplasm, nuclear regions, and vacuoles limited by membranes. Small bodies often were present inside the vacuoles. These bodies also contained a peripheral membrane and a granular cytoplasm but usually no nuclear regions. The normal bacteria from which the L forms were derived differed markedly in structure from one another, especially in the surface layers of the cells.     

Microbial interactions involving sulfur bacteria: implications for the ecology and evolution of bacterial communities

A major goal of microbial ecology is the identification and characterization of those microorganisms which govern transformations in natural ecosystems. This review summarizes our present knowledge of microbial interactions in the natural sulfur cycle. Central to the discussion is the recent progress made in understanding the co-occurrence in natural ecosystems of sulfur bacteria with contrasting nutritional requirements and of the spatially very close associations of bacteria, the so-called phototrophic consortia (e.g. 'Chlorochromatium aggregatum' or 'Pelochromatium roseum'). In a similar way, microbial interactions may also be significant during microbial transformations other than the sulfur cycle in natural ecosystems, and could also explain the low culturability of bacteria from natural samples.     

Analytical strategies for the global quantification of intact proteins

The quantification of intact proteins is a relatively recent development in proteomics. In eukaryotic organisms, proteins are present as multiple isoforms as the result of variations in genetic code, alternative splicing, post-translational modification and other processing events. Understanding the identities and biological functions of these isoforms and how their concentrations vary across different states is the central goal of proteomics. To date, the bulk of proteomics research utilizes a "bottom-up" approach, digesting proteins into their more manageable constitutive peptides, but sacrificing information about the specific isoform and combinations of post-translational modifications present on the protein. Very specific strategies for protein quantification such as the enzyme-linked immunosorbent assay and Western blot are commonplace in laboratories and clinics, but impractical for the study of global biological changes. Herein, we describe strategies for the quantification of intact proteins, their distinct advantages, and challenges to their employment. Techniques contained in this review include the more traditional and widely employed methodology of differential gel electrophoresis and more recently developed mass spectrometry-based techniques including metabolic labeling, chemical labeling, and label-free methodologies.     

Soluble bacterial constituents down-regulate secretion of IL-12 in response to intact Gram-positive bacteria

Intact Gram-positive bacteria induce production of large amounts of IL-12 from freshly isolated human monocytes. Here the bacterial structures and signalling pathways involved were studied and compared with those leading to IL-6 production, and to IL-12 production in response to LPS after IFN-gamma pre-treatment. Intact bifidobacteria induced massive production of IL-12 (1 ng/ml) and IL-6 (>30 ng/ml) from human PBMC, whereas fragmented bifidobacteria induced IL-6, but no IL-12. IL-12 production induced by intact bifidobacteria was inhibited by pre-treatment with bifidobacterial sonicate, peptidoglycan, muramyl dipeptide, lipoteichoic acid, the soluble TLR2 agonist Pam(3)Cys-SK(4), or anti-TLR2 antibodies. Blocking of phagocytosis by cytochalasin, inhibition of the JNK or NF-kappaB pathways or treatment with Wortmannin also reduced the IL-12 response to intact Gram-positive bacteria. LPS induced moderate levels of IL-12 (0.31 ng/ml), but only from IFN-gamma pre-treated PBMC. This IL-12 production was enhanced by Wortmannin and unaffected by blocking the JNK pathway. Thus, intact Gram-positive bacteria trigger monocyte production of large amounts of IL-12 via a distinct pathway that is turned off by fragmented Gram-positive bacteria. This may be a physiological feedback, since such fragments may signal that further activation of the phagocyte via the IL-12/IFN-gamma loop is unnecessary.     

Assay for the quantification of intact/fragmented genomic DNA

This study shows that the accuracy of the quantification of genomic DNA by the commonly used Hoechst- and PicoGreen-based assays is drastically affected by its degree of fragmentation. Specifically, it was shown that these assays underestimate by 70% the concentration of double-stranded DNA (dsDNA) with sizes less than 23 kb. On the other hand, DNA sizes greater and less than approximately 23 kb are commonly characterized as intact and fragmented genomic DNA, respectively, by the agarose electrophoresis DNA smearing assay and are evaluated only qualitatively by this assay. The need for accurate quantification of fragmented and total genomic DNA, combined with the lack of specific, reliable, and simple quantitative methods, prompted us to develop a Hoechst/PicoGreen-based fluorescent assay that quantifies both types of DNA. This assay addresses these problems, and in its Hoechst and PicoGreen version it accurately quantifies dsDNA as being either intact (>or=23 kb) or fragmented (<23 kb) in concentrations as low as 3 ng ml-1 or 5 pg ml-1 with Hoechst or PicoGreen, respectively, as well as the individual fractions of intact/fragmented DNA existing in any proportions in a total DNA sample in concentrations as low as 10 ng ml-1 or 15 pg ml-1 with Hoechst or PicoGreen, respectively. Because the assay discriminates total genomic DNA in the two size ranges (>or=23 and <23 kb) and quantitates them, it is proposed as the quantitative replacement of the agarose gel electrophoresis genomic DNA smearing assay.     

Synthesis of novel isoluminol probes and their use in rapid bacterial assays

Rapid assays for bacteria have been developed utilizing novel LysLysLys-isoluminol (14) and GluGlu-isoluminol (16) probes that have been derived from peptides which potentially mimic bacteriophage attachment protein binding regions. Compared to two conventional methods that are widely used, namely nucleic acid probes and polymerase chain reaction (PCR) assays, these types of probes may eventually have certain advantages, such as high sensitivity, and short preparation and assay time.     

古菌和细菌四醚膜脂GDGTs的生物合成机制及其生物地球化学意义

以甘油二烷基甘油四醚(glycerol dialkyl glycerol tetraethers,GDGTs)为主的跨膜醚脂化合物是古菌和部分细菌细胞膜的重要组成成分。作为分子化石,GDGTs化合物对环境变化响应敏感,以其为基础的有机地球化学指标在定量重建海洋与陆地的古环境研究中发挥出独特的优势。然而,GDGTs指标在广泛应用的同时也不断出现适用性和准确性问题。其关键原因在于GDGTs的生物合成和生理机制研究相对匮乏,难以为指标提供分子生物学与生理学基础。近年来,在多学科的交叉融合下,古菌类异戊二烯GDGTs的生物合成研究取得了令人瞩目的进展。这些成果为脂类生物标志物的地学应用及生物源的确定提供了可靠的生物学基础和新的研究思路。本文综述了古菌类异戊二烯GDGTs的生物合成过程,提出了细菌支链GDGTs生物合成途径的假说,讨论了GDGTs生理过程的生物地球化学意义,并初步展望了GDGTs研究领域未来重要的发展方向。     

Superantigens of gram-positive bacteria: structure-function analyses and their implications for biological activity

Just as we thought that we know everything about superantigens, new molecular and structural studies indicate that we have only just begun to unravel the secrets of these fascinating molecules. Recent structure-function analysis of superantigens from Gram-positive bacteria, with emphasis on their interaction with major histocompatibility complex molecules, could help us decipher the role of superantigens in disease, identify host factors that potentiate their effects and design drugs that specifically block their activity.     

Peptidoglycan types of bacterial cell walls and their taxonomic implications

[This corrects the article on p. 409 in vol. 36.].