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PubMed2003

Origins of highly mosaic mycobacteriophage genomes.

Bacteriophages are the most abundant organisms in the biosphere and play major roles in the ecological balance of microbial life. The genomic sequences of ten newly isolated mycobacteriophages suggest that the bacteriophage population as a whole is amazingly diverse and may represent the largest unexplored reservoir of sequence information in the biosphere. Genomic comparison of these mycobacteriophages contributes to our understanding of the mechanisms of viral evolution and provides compelling evidence for the role of illegitimate recombination in horizontal genetic exchange. The promiscuity of these recombination events results in the inclusion of many unexpected genes including those implicated in mycobacterial latency, the cellular and immune responses to mycobacterial infections, and autoimmune diseases such as human lupus. While the role of phages as vehicles of toxin genes is well established, these observations suggest a much broader involvement of phages in bacterial virulence and the host response to bacterial infections.

Autoimmune Diseases↗
PubMed2000

Attenuation mechanisms of N-nitrosodimethylamine at an operating intercept and treat groundwater remediation system.

The North Boundary Containment System (NBCS), an intercept-and-treat system, was established at Rocky Mountain Arsenal (RMA), Commerce City, CO, to remove low-level organic contaminants from a groundwater plume exiting RMA to the north and northwest. N-nitrosodimethylamine (NDMA) was detected in groundwater collected from the dewatering and recharge zones of the NBCS system. Concern over the fate of NDMA, in terms of potentially exiting the boundaries of the arsenal, prompted an investigation to evaluate potential attenuation mechanisms for NDMA within the alluvial aquifer system and within the NBCS itself. Groundwater, soil, and granular activated carbon (GAC) samples were taken from key locations in the NBCS system. Soil and GAC samples were assayed for sorption kinetics and for adsorption and desorption properties using 14C-labeled NDMA. NDMA biodegradation experiments were conducted by following 14CO(2) evolution from 14C-labeled NDMA in soils and GAC samples under aerobic and anaerobic conditions. The sorptive capacity of the site soils for NDMA was insignificant. Furthermore, the adsorption of the NDMA by the soil was almost completely reversible. Evaluation of the degradation potential of the native microbial consortia indicated a high level of NDMA mineralization when measured using bench-scale microcosms. The native consortia had capability to mineralize the NDMA under both aerobic and anaerobic incubations, indicating facultative characteristics. Testing of the local groundwater chemistry revealed that the area of the aquifer of interest was microaerobic and neutral in pH. These conditions were optimal for NDMA removal. While sorption was insignificant, degradation was a significant attenuation mechanism, which may be the reason that no NDMA has migrated off-site. This gives rise to the potential of a long-term sink for attenuating NDMA within the recharge zone of the treatment system.

Adsorption↗
PubMed2000

Genome data: what do we learn?

Genome sequence information has continued to accumulate at a spectacular pace during the past year. Details of the sequence and gene content of human chromosome 22 were published. The sequencing and annotation of the first two Arabidopsis thaliana chromosomes was completed. The sequence of chromosome 3 from Plasmodium falciparum, the second sequenced malaria chromosome, was reported, as was that of chromosome 1 from Leishmania major. The complete genomic sequences of five microbes were reported. Approaches to using data from completely sequenced microbial genomes in phylogenetic studies are being explored, as is the application of microarrays to whole genome expression analysis.

Animals↗
PubMed2002

Drosophila innate immunity: an evolutionary perspective.

In response to microbial infections, Drosophila mounts a multifaceted immune response involving humoral reactions that culminate in the destruction of invading organisms by lytic peptides. These defense mechanisms are activated via two distinct signaling pathways. One of these, the Toll pathway, controls resistance to fungal and Gram-positive bacterial infections, whereas the Imd pathway is responsible for defense against Gram-negative bacterial infections. Current evidence indicates that recognition of infectious nonself agents results from interactions between microbial wall components and extracellular pattern recognition proteins. We discuss here evolutionary perspectives on our present understanding of the antimicrobial defenses of Drosophila.

Animals↗
PubMed1994

Biodiversity at the molecular level: the domains, kingdoms and phyla of life.

The results of comparative sequence analysis, mainly of small subunit (SSU) ribosomal (r)RNA sequences, have suggested that all of cellular life can be placed in one of three domains: the Archaea, Bacteria or Eucarya. There is some evidence that the Archaea may not be a monophyletic assemblage, but as yet this issue has not been resolved. Most of the lineages, and all of the deepest ones, in the tree based upon SSU rRNA sequences, are microbial. Traditional ideas of classification such as Whittaker's five kingdom scheme do not adequately describe life's diversity as revealed by sequence comparisons. There are many microbial groups that demonstrate much greater amounts of SSU rRNA sequence divergence than do members of the classical kingdoms, Animalia, Plantae and Fungi. The old microbial kingdoms Monera and Protista are clearly paraphyletic but as yet there is no consensus as to how they should be reorganized in taxonomic terms. New data from environmental analysis suggests that much of the microbial world is unknown. Every environment which has been analysed by molecular methods has revealed many previously unrecorded lineages. Some of these show great divergence from the sequences of cultured microorganisms suggesting that fundamentally new microbial groups remain to be isolated. The relationships of some of these new lineages may be expected to affect how the tree of life is organized into higher taxa, and to also influence which features will be recognized as synapomorphies. There is currently no objective measure whereby microbial diversity can be quantified and compared to the figures which are widely quoted for arthropods and other Metazoa.

Animals↗
PubMed2006

Implications of a 3.472-3.333 Gyr-old subaerial microbial mat from the Barberton greenstone belt, South Africa for the UV environmental conditions on the early Earth.

Modelling suggests that the UV radiation environment of the early Earth, with DNA weighted irradiances of about three orders of magnitude greater than those at present, was hostile to life forms at the surface, unless they lived in specific protected habitats. However, we present empirical evidence that challenges this commonly held view. We describe a well-developed microbial mat that formed on the surface of volcanic littoral sediments in an evaporitic environment in a 3.5-3.3Ga-old formation from the Barberton greenstone belt. Using a multiscale, multidisciplinary approach designed to strongly test the biogenicity of potential microbial structures, we show that the mat was constructed under flowing water by 0.25 microm filaments that produced copious quantities of extracellular polymeric substances, representing probably anoxygenic photosynthesizers. Associated with the mat is a small colony of rods-vibroids that probably represent sulphur-reducing bacteria. An embedded suite of evaporite minerals and desiccation cracks in the surface of the mat demonstrates that it was periodically exposed to the air in an evaporitic environment. We conclude that DNA-damaging UV radiation fluxes at the surface of the Earth at this period must either have been low (absorbed by CO2, H2O, a thin organic haze from photo-dissociated CH4, or SO2 from volcanic outgassing; scattered by volcanic, and periodically, meteorite dust, as well as by the upper layers of the microbial mat) and/or that the micro-organisms exhibited efficient gene repair/survival strategies.

Bacteria↗
PubMed2004

Morphological and physiological changes of Staphylococcus aureus exposed to hypochlorous acid.

AIMS: To characterize hypochlorous acid (HOCl) stress resistance of Staphylococcus aureus and to assess physiological state and changes in cell morphology. METHODS AND RESULTS: Clinical wild-type strain of S. aureus was used in the stress with HOCl at concentrations ranging from 0 to 4 mg l(-1). Concentrations below 1.5 mg l(-1) caused no significant drop in viability. During 2 h of HOCl stress at 2 mg l(-1), there was appearance of minicells capable of passing through the 0.45 microm pores of filtration membranes. Intracellular proteins increased gradually to reach a level of 51% of dry weight and an enhanced synthesis of at least two proteins of 23 and 220 kDa was concluded. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Staphylococcus aureus can undergo morphological and physiological changes during 2 h of exposure to 2 mg l(-1) of HOCl, which represents an adaptative response towards the hypochlorous acid stress. This evolution limits the use of 0.45 microm pores size membrane filters for research on S. aureus in waters and the clinical environment.

Bacterial Proteins↗
PubMed2004

Low-pass sequencing for microbial comparative genomics.

BACKGROUND: We studied four extremely halophilic archaea by low-pass shotgun sequencing: (1) the metabolically versatile Haloarcula marismortui; (2) the non-pigmented Natrialba asiatica; (3) the psychrophile Halorubrum lacusprofundi and (4) the Dead Sea isolate Halobaculum gomorrense. Approximately one thousand single pass genomic sequences per genome were obtained. The data were analyzed by comparative genomic analyses using the completed Halobacterium sp. NRC-1 genome as a reference. Low-pass shotgun sequencing is a simple, inexpensive, and rapid approach that can readily be performed on any cultured microbe. RESULTS: As expected, the four archaeal halophiles analyzed exhibit both bacterial and eukaryotic characteristics as well as uniquely archaeal traits. All five halophiles exhibit greater than sixty percent GC content and low isoelectric points (pI) for their predicted proteins. Multiple insertion sequence (IS) elements, often involved in genome rearrangements, were identified in H. lacusprofundi and H. marismortui. The core biological functions that govern cellular and genetic mechanisms of H. sp. NRC-1 appear to be conserved in these four other halophiles. Multiple TATA box binding protein (TBP) and transcription factor IIB (TFB) homologs were identified from most of the four shotgunned halophiles. The reconstructed molecular tree of all five halophiles shows a large divergence between these species, but with the closest relationship being between H. sp. NRC-1 and H. lacusprofundi. CONCLUSION: Despite the diverse habitats of these species, all five halophiles share (1) high GC content and (2) low protein isoelectric points, which are characteristics associated with environmental exposure to UV radiation and hypersalinity, respectively. Identification of multiple IS elements in the genome of H. lacusprofundi and H. marismortui suggest that genome structure and dynamic genome reorganization might be similar to that previously observed in the IS-element rich genome of H. sp. NRC-1. Identification of multiple TBP and TFB homologs in these four halophiles are consistent with the hypothesis that different types of complex transcriptional regulation may occur through multiple TBP-TFB combinations in response to rapidly changing environmental conditions. Low-pass shotgun sequence analyses of genomes permit extensive and diverse analyses, and should be generally useful for comparative microbial genomics.

Archaea↗
PubMed1999

Rapid direct methods for enumeration of specific, active bacteria in water and biofilms.

Conventional methods for detecting indicator and pathogenic bacteria in water may underestimate the actual population due to sublethal environmental injury, inability of the target bacteria to take up nutrients and other physiological factors which reduce bacterial culturability. Rapid and direct methods are needed to more accurately detect and enumerate active bacteria. Such a methodological advance would provide greater sensitivity in assessing the microbiological safety of water and food. The principle goal of this presentation is to describe novel approaches we have formulated for the rapid and simultaneous detection of bacteria plus the determination of their physiological activity in water and other environmental samples. The present version of our method involves the concentration of organisms by membrane filtration or immunomagnetic separation and combines an intracellular fluorochrome (CTC) for assessment of respiratory activity plus fluorescent-labelled antibody detection of specific bacteria. This approach has also been successfully used to demonstrate spatial and temporal heterogeneities of physiological activities in biofilms when coupled with cryosectioning. Candidate physiological stains include those capable of determining respiratory activity, membrane potential, membrane integrity, growth rate and cellular enzymatic activities. Results obtained thus far indicate that immunomagnetic separation can provide a high degree of sensitivity in the recovery of seeded target bacteria (Escherichia coli O157:H7) in water and hamburger. The captured and stained target bacteria are then enumerated by either conventional fluorescence microscopy or ChemScan(R), a new instrument that is very sensitive and rapid. The ChemScan(R) laser scanning instrument (Chemunex, Paris, France) provides the detection of individual fluorescently labelled bacterial cells using three emission channels in less than 5 min. A high degree of correlation has been demonstrated between results obtained with the ChemScan and traditional plate counts of mixed natural bacterial populations in water. The continuing evolution of these methods will be valuable in the rapid and accurate analysis of environmental samples.

Bacteria↗
PubMed2005

Molecular characterization of TEM-type beta-lactamases identified in cold-seep sediments of Edison Seamount (south of Lihir Island, Papua New Guinea).

To determine the prevalence and genotypes of beta-lactamases among clones of a metagenomic library from the cold-seep sediments of Edison seamount (10,000 years old), we performed pulse-field gel electrophoresis, antibiotic susceptibility testing, pI determination, and DNA sequencing analysis. Among the 8,823 clones of the library, thirty clones produced beta-lactamases and had high levels of genetic diversity. Consistent with minimum inhibitory concentration patterns, we found that five (16.7%) of thirty clones produced an extended-spectrum beta-lactamase. 837- and 259-bp fragments specific to blaTEM genes were amplified, as determined by banding patterns of PCR amplification with designed primers. TEM-1 was the most prevalent beta-lactamase and conferred resistance to ampicillin, piperacillin, and cephalothin. TEM-116 had a spectrum that was extended to ceftazidime, cefotaxime, and aztreonam. The resistance levels conferred by the pre-antibiotic era alleles of TEM-type beta-lactamases were essentially the same as the resistance levels conferred by the TEM-type alleles which had been isolated from clinically resistant strains of bacteria of the antibiotic era. Our first report on TEM-type beta-lactamases of the pre-antibiotic era indicates that TEM-type beta-lactamases paint a picture in which most of the diversity of the enzymes may not be the result of recent evolution, but that of ancient evolution.

Gene Library↗
PubMed1984

Preparation and sterilization by filtration of Renacidin irrigation.

A method for the sterilizing filtration of Renacidin, a urologic irrigating solution, was evaluated. Renacidin irrigation was prepared and sterilized by microporous membrane filtration. A sterilizing membrane filtration apparatus was challenged by inoculating a batch of irrigation solution with Escherichia coli. The sterility of both intentionally contaminated and routinely prepared batches was evaluated. The stability of the solution was monitored by pH measurement, visual examination, maintenance of a vacuum, and absorbance spectrum of a 1:100 dilution in deionized water over a wavelength range from 400 to 200 nm. The time required to prepare three one-liter units was about two hours. No microbial growth was detected in any of the samples. The predicted minimum shelf-life at 10 degrees C was six months. Because the prepared solution contains some unreacted citric acid and bicarbonates, storage at room temperature could produce excessive pressure inside the container from carbon dioxide gas evolution. Refrigerated storage is recommended. This method for the preparation and sterilization of Renacidin irrigation is reasonably expedient, economical, and reliable.

Chemical Phenomena↗
PubMed2001

On the need for another type of predictive model in structured foods.

Most of the models discussed up till now in predictive microbiology do not take into account the variability of microbial growth with respect to space. In structured (solid) foods, microbial growth can strongly depend on the position in the food and the assumption of homogeneity can thus not be accepted: space must be considered as an independent variable. Indeed, experimental evidence exists of bacteria competition on agar not showing the same behavior as the competition in a well-mixed liquid culture system. It is conjectured that this is due to the spatially structured habitat. Therefore, in the current paper, a prototype two species competition model proposed in previous work by the authors is extended to take space into account. The extended model describes two phenomena: (i) local evolution of biomass and (ii) transfer of biomass through the medium. The structure of the food product is taken into account by limiting the diffusion through the medium. The smaller mobility of the micro-organisms in solid foods allows spatial segregation which causes pattern formation. Evidence is given for the fact that taking space into account indeed has an influence on the behavior (coexistence/extinction) of the populations. Although the reported simulations are by no means to be interpreted as accurate predictions, the proposed model structure allows one to highlight (i) important characteristics of microbial growth in structured foods and (ii) future research trends in predictive microbiology.

Bacteria↗
PubMed2004

Evolution of the PPM-family protein phosphatases in Streptomyces: duplication of catalytic domain and lateral recruitment of additional sensory domains.

Originally identified from eukaryotes, the Mg2+- or Mn2+-dependent protein phosphatases (PPMs) are a diverse group of enzymes whose members include eukaryotic PP2C and some prokaryotic serine/threonine phosphatases. In a previous study, unexpectedly large numbers of PPMs were identified in two Streptomyces genomes. In this work, a phylogenetic analysis was performed with all the PPMs available from a wide variety of microbial sources to determine the evolutionary origin of the Streptomyces PPM proteins. Consistent with earlier hypotheses, the results suggested that the microbial PPMs were relatively recent additions from eukaryotic sources. Results also indicated that the Streptomyces PPMs were divided into two major subfamilies at an early stage of their emergence in Streptomyces genomes. The first subfamily, which contains only six Streptomyces PPMs, possesses a catalytic domain whose sequence and architecture are similar to that of eukaryotic PPMs; the second subfamily contains 89 Streptomyces PPMs that lack the 5a and 5b catalytic domain motifs, similar to the PPMs SpoIIE and RsbU of Bacillus subtilis. Significant gene duplication was observed for the PPMs in the second subfamily. In addition, more than half (54 %) of the Streptomyces PPMs from the second subfamily were found to have at least one additional sensory domain, most commonly the PAS or the GAF domain. Phylogenetic analysis showed that these domains tended to be clustered according to the putative physiological functions rather than taxonomic relationship, implying that they might have arisen as a result of domain recruitment in a late evolutionary stage. This study provides an insight into how Streptomyces spp. may have expanded their PPM-based signal transduction networks to enable them to respond to a greater range of environmental changes.

Catalytic Domain↗
PubMed2003

Genome-scale microbial in silico models: the constraints-based approach.

Genome sequencing and annotation has enabled the reconstruction of genome-scale metabolic networks. The phenotypic functions that these networks allow for can be defined and studied using constraints-based models and in silico simulation. Several useful predictions have been obtained from such in silico models, including substrate preference, consequences of gene deletions, optimal growth patterns, outcomes of adaptive evolution and shifts in expression profiles. The success rate of these predictions is typically in the order of 70-90% depending on the organism studied and the type of prediction being made. These results are useful as a basis for iterative model building and for several practical applications.

Animals↗
PubMed1997

Resistance patterns of streptococcus pneumoniae from carriers attending day-care centers in southwestern Greece.

The resistance to beta-lactam and non-beta-lactam antibiotics of 133 nasopharyngeal isolates of Streptococcus pneumoniae recovered from December 1995 to February 1996 from children attending seven day-care centers in southwestern Greece was studied. Reduced susceptibility to one or more anti-microbial agents was found in 70 isolates (53%), as follows: penicillin, 17% intermediate, 12% resistant; cefotaxime, 10.5% intermediate, 1.5% resistant; trimethoprim-sulfamethoxazole, 8% intermediate, 35% resistant; chloramphenicol, 27% resistant; tetracycline, 29% resistant; and erythromycin/clindamycin, 19% resistant. Eighty-seven percent of penicillin-intermediate or -resistant strains belonged to serogroups/serotypes 19, 21, and 23. Fifty-six percent of the antibiotic-resistant pneumococci were multiply resistant, including serogroup 6 strains that were penicillin-susceptible but resistant to all non-beta-lactam drugs tested, as well as serogroup 23 strains resistant to penicillin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole. The high incidence of antibiotic-resistant pneumococci and the divergent and unique resistance patterns found in this study underline the need for global surveillance of S. pneumoniae to document the evolution and spread of resistant strains and to guide therapy.

Anti-Bacterial Agents↗
PubMed2003

Mitochondria and hydrogenosomes are two forms of the same fundamental organelle.

Published data suggest that hydrogenosomes, organelles found in diverse anaerobic eukaryotes that make energy and hydrogen, were once mitochondria. As hydrogenosomes generally lack a genome, the conversion is probably one way. The sources of the key hydrogenosomal enzymes, pyruvate : ferredoxin oxidoreductase (PFO) and hydrogenase, are not resolved by current phylogenetic analyses, but it is likely that both were present at an early stage of eukaryotic evolution. Once thought to be restricted to a few unusual anaerobic eukaryotes, the proteins are intimately integrated into the fabric of diverse eukaryotic cells, where they are targeted to different cell compartments, and not just hydrogenosomes. There is no evidence supporting the view that PFO and hydrogenase originated from the mitochondrial endosymbiont, as posited by the hydrogen hypothesis for eukaryogenesis. Other organelles derived from mitochondria have now been described in anaerobic and parasitic microbial eukaryotes, including species that were once thought to have diverged before the mitochondrial symbiosis. It thus seems possible that all eukaryotes may eventually be shown to contain an organelle of mitochondrial ancestry, to which different types of biochemistry can be targeted. It remains to be seen if, despite their obvious differences, this family of organelles shares a common function of importance for the eukaryotic cell, other than energy production, that might provide the underlying selection pressure for organelle retention.

Anaerobiosis↗
PubMed2005

Influence of bulking agents and microbial activator on thermophilic aerobic transformation of sewage sludge.

Bangkok, while improving the wastewater treatment in order to alleviate the river pollution, faces important amounts of sewage sludge. The sewage sludge contains organic matter, nitrogen and phosphorus available for plant growth. However, it may contain pathogenic microorganisms. To be used for agricultural purposes, these pathogens should be destroyed, which can be achieved with the thermophilic phase of composting. As the sewage sludge is dense and unable to compost alone (low C/N ratio), it should be mixed with an organic by-product. Two by-products available in large quantities in Thailand (wood chips and rice husk) have been tested for mixture with sewage sludge. As these products are not easy to decompose (presence of silica in rice husk and lignin/tannins in wood chips), the addition of a microbial activator for composting has been tested in controlled conditions (small quantities of organic mixtures, 55 degrees C, moisture maintained at 60-70% of water holding capacity). The monitoring of the decomposition has been made by measuring the carbon dioxide respiration, pH, organic matter and nitrogen contents and the evolution of enzymatic activities. When mixed with sewage sludge, wood chips and rice husk do not show significant differences concerning decomposition after 63 days. The use of an activator within the experimental conditions does not improve the decomposition of organic matter contained in the mixture of sewage sludge and rice husk or wood chips.

Analysis of Variance↗