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PubMed1985

Physiological characterization of adaptive clones in evolving populations of the yeast, Saccharomyces cerevisiae.

Populations of a diploid strain of S. cerevisiae were grown in glucose-limited continuous culture for more than 260 generations. A series of seven sequential adaptive changes were identified by monitoring the frequency of cycloheximide resistance in these populations. Samples were taken from the continuous cultures following each adaptive shift and characterized physiologically to determine (1) the range of phenotypes that can be selected in a precisely defined constant environment and (2) the order and predictability of the occurrence of the adaptive mutations in evolving populations. The clones were characterized with respect to the growth parameters, maximum growth rate, saturation coefficient and yield, as well as for changes in cell size and geometry and rate of glucose uptake. The maximum growth rates of the seven adaptive clones were very similar, but in contrast the saturation coefficients differed substantially. Surprisingly, not all clones showed reductions in the saturation coefficients, in comparison to the immediately preceding clones, as would be predicted from classical continuous culture kinetics. In addition, yield estimates first increased and then decreased for later isolated adaptive clones. In general, the results suggest epistatic interactions between the adaptive clones, consistent with earlier published results. The rate of glucose uptake, as measured by 14C-xylose uptake, increased dramatically after the selection and fixation of seven adaptive clones. Progressive decreases in cell volume and changes in cell geometry, resulting in increased surface area to volume ratios, were also observed in the adaptive clones, but these changes were not always seen in other haploid and diploid yeast populations evolving under the same conditions. Such changes may be easily explainable in terms of the characteristics of the glucose-limited environment. The significance of the results to the evolution of microorganisms under nutrient-limiting conditions is discussed.

Biological Transport↗
PubMed1998

A chromosomal ars operon homologue of Pseudomonas aeruginosa confers increased resistance to arsenic and antimony in Escherichia coli.

Operons encoding homologous arsenic-resistance determinants (ars) have been discovered in bacterial plasmids from Gram-positive and Gram-negative organisms, as well as in the Escherichia coli chromosome. However, evidence for this arsenic-resistance determinant in the medically and environmentally important bacterial species Pseudomonas aeruginosa is conflicting. Here the identification of a P. aeruginosa chromosomal ars operon homologue via cloning and complementation of an E. coli ars mutant is reported. The P. aeruginosa chromosomal ars operon contains three potential ORFs encoding proteins with significant sequence similarity to those encoded by the arsR, arsB and arsC genes of the plasmid-based and E. coli chromosomal ars operons. The cloned P. aeruginosa chromosomal ars operon confers augmented resistance to arsenic and antimony oxyanions in an E. coli arsB mutant and in wild-type P. aeruginosa. Expression of the operon was induced by arsenite at the mRNA level. DNA sequences homologous with this operon were detected in some, but not all, species of the genus Pseudomonas, suggesting that its conservation follows their taxonomic-based evolution.

Adenosine Triphosphatases↗
PubMed2003

Geographic barriers isolate endemic populations of hyperthermophilic archaea.

Barriers to dispersal between populations allow them to diverge through local adaptation or random genetic drift. High-resolution multilocus sequence analysis revealed that, on a global scale, populations of hyperthermophilic microorganisms are isolated from one another by geographic barriers and have diverged over the course of their recent evolutionary history. The identification of a biogeographic pattern in the archaeon Sulfolobus challenges the current model of microbial biodiversity in which unrestricted dispersal constrains the development of global species richness.

Analysis of Variance↗
PubMed1992

Characterization of In0 of Pseudomonas aeruginosa plasmid pVS1, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria.

Many multiresistance plasmids and transposons of gram-negative bacteria carry related DNA elements that appear to have evolved from a common ancestor by site-specific integration of discrete cassettes containing antibiotic resistance genes or sequences of unknown function. The site of integration is flanked by conserved segments coding for an integraselike protein and for sulfonamide resistance, respectively. These segments, together with the antibiotic resistance genes between them, have been termed integrons (H. W. Stokes and R. M. Hall, Mol. Microbiol. 3:1669-1683, 1989). We report here the characterization of an integron, In0, from Pseudomonas aeruginosa plasmid pVS1, which has an unoccupied integration site and hence may be an ancestor of more complex integrons. Codon usage of the integrase (int) and sulfonamide resistance (sul1) genes carried by this integron suggests a common origin. This contrasts with the codon usage of other antibiotic resistance genes that were presumably integrated later as cassettes during the evolution and spread of these DNA elements. We propose evolutionary schemes for (i) the genesis of the integrons by the site-specific integration of antibiotic resistance genes and (ii) the evolution of the integrons of multiresistance plasmids and transposons, in relation to the evolution of transposons related to Tn21.

Amino Acid Sequence↗
PubMed2000

Molecular mechanisms of innate immunity.

All species require a rapid, systemic reply to pathogens in their environment. This response is known as the innate immune response and is characterized by de novo synthesis of mediators that directly or indirectly through phagocytosis remove and kill the pathogen. Innate immune responses have been preserved throughout evolution and have been studied in detail in organisms from the fruit fly Drosophila melanogaster to humans. In my laboratory, studies performed during the past 25 yr have focused on defining the molecular basis of innate immune responses to microbial pathogens. Specifically, we have used bacterial endotoxin (lipopolysaccharide) as a model stimulus to define how the innate immune system recognizes products of microbial pathogens and initiates responses to remove and/or kill such organisms. Such studies also serve as models to understand more fully the mechanisms underlying a serious human disease known as septic shock. This article discusses septic shock and its relationship to innate immunity.

Animals↗
PubMed2000

Integrons: an antibiotic resistance gene capture and expression system.

Bacteria can transfer genetic information to provide themselves with protection against most antibiotics. The acquisition of resistance gene arrays involves genetic mobile elements like plasmids and transposons. Another class of genetic structures, termed integrons, have been described and contain one or more gene cassettes located at a specific site. Integrons are defined by an intl gene encoding an integrase, a recombination site attl and a strong promoter. At least six classes of integrons have been determined according to their intl gene. Classes 1, 2 and 3 are the most studied and are largely implicated in the dissemination of antibiotic resistance. A gene cassette includes an open reading frame and, at the 3'-end, a recombination site attC. Integration or excision of cassettes occur by a site-specific recombination mechanism catalyzed by the integrase. However, insertion can occur, albeit rarely, at non-specific sites leading to a stable situation for the cassette. Cassettes are transcribed from the common promoter located in the 5'-conserved segment and expression of distal genes is reduced by the presence of upstream cassettes. Most gene cassettes encode antibiotic resistant determinants but antiseptic resistant genes have also been described. Integrons seem to have a major role in the spread of multidrug resistance in gram-negative bacteria but integrons in gram-positive bacteria were described recently. Moreover, the finding of super-integrons with gene-cassettes coding for other determinants (biochemical functions, virulence factors) in Vibrio isolates dating from 1888 suggests the likely implication of this multicomponent cassette-integron system in bacterial genome evolution before the antibiotic era and to a greater extent than initially believed.

Animals↗
PubMed2000

[Pharmacokinetic effects of antibiotics on the development of bacterial resistance particularly in reference to azithromycin].

Antibiotics reduce the mortality from infectious diseases but not the prevalence of these diseases. Use, and often abuse, of antimicrobial agents encourages the evolution of bacteria toward resistance, often resulting in therapeutic failure. There are two factors which influence potential utility of a drug in a specific clinical situation. The first is the measure of potency of the antibiotic for the pathogen in question (minimal inhibitory concentration [MIC], minimal bactericidal concentration [MBC]). The second is whichever relationship between the concentration-time profile and potency of the antibiotic linked most robustly to clinical outcome (time above MIC or MBC [T > MIC or T > MBC]; Peak/MIC or MBC; area under the curve [AUC]/MIC or AUC/MBC). Herein the effects of pharmacokinetics of antimicrobials on the evolution of antimicrobial resistance with particular reference to azithromycin are considered.

Aminoglycosides↗
PubMed1998

[Bacteremia caused by Stenotrophomonas maltophilia: a clinical-epidemiological study and resistance profile].

Over a 7-year period (1990-1996), the causal disease, predisposing factors, focus infection, clinical manifestations, complications and evolution of patients presenting bacteremia from Stenotrophomonas maltophilia were analyzed retrospectively in a university hospital. A microbiological study was carried out to determine the percentage of positive blood cultures per episode and the characteristics of bacteremia and to evaluate the antibiotic susceptibility of the isolated strains. Twenty-seven episodes of bacteremia from S. maltophilia were identified in 26 patients, half of whom were women, and the median age was 40 years. A total of 48% of the patients had blood malignancy (12 cases), with acute myeloid leukemia (5 cases) being the most frequent. Seven patients (27%) needed to be admitted to an intensive care unit for some type of vital support and/or intensive treatment. The previous administration of large spectrum antimicrobials (21/26) and the presence of a catheter or central catheter (19/26) were the most frequently found predisposing factors (81% and 73%, respectively). One-quarter of the patients had received treatment with carbapenems. Immunodepression caused by chemotherapy or corticosteroids occurred in 65% of the cases. Half of the patients had undergone a major surgical procedure or had been intubated and submitted to mechanical ventilation. One-third presented granulocytopenia on the detection of bacteremia and 6 of the 12 patients with blood malignancy showed severe neutropenia (<500 neutrophils/mm3). The bacteremia was acquired in hospital in 78% of the cases (21/27). In 26% of these cases, S. maltophilia was diagnosed as the probable cause of bacteremia; in 34% this was just a possibility. The origin of the bacteremia was unknown in 11 cases (40%). Infection from the vascular catheter was the most frequent focus infection (7 cases). An average of 3.6 blood cultures were performed per patient, out of a total of 92, and 54% showed positive. The average time of growth for S. maltophilia in the blood culture bottles was 30 hours. One-third of the bacteremia episodes from S. maltophilia were polymicrobial (9/27). Clinical evolution was favorable in 18 patients, while 8 died (31%), 5 cases (20%) from causes directly related to the bacteremia (4 from septic shock). The mortality associated with the polymicrobial bacteremia was not significantly different from that for single microbial bacteremia from S. maltophilia. Ninety percent of the isolated strains showed susceptibility to co-trimoxazole, 77% to ticarcillin-clavulanic acid, 60% to ciprofloxacin, 62% to ceftazidime, 20% to amikacin and just 18% to imipenem.

Adolescent↗
PubMed2001

Microbial survivors: thermophiles, halophiles, and other prodigies.

When biologists encounter microbes flourishing in boiling water and other extreme habitats, they often consider such creatures as merely odd, and only search for possible protective mechanisms. But it may also be that extreme habitats resemble those where life first occurred, and that such organisms provide links with earlier evolution.

Cyanobacteria↗
PubMed2001

In search of natural substrates and inhibitors of MDR pumps.

The function of microbial MDRs remains a hotly debated subject. Given the very broad substrate specificities of some MDRs, like the RND pumps that can extrude all classes of amphipathic compounds (cationic, neutral, and anionic), it seems difficult to develop a rationale for pinpointing possible natural substrates of these translocases. At the same time, several clues can be used to guide our search for natural MDR substrates. One is the fact that amphipathic cations appear to be the preferred substrates of MDRs. These substances are extruded by MDRs of all 5 known families and are the almost exclusive substrates of SMR and MF family MDRs. The universal nature of amphipathic cations as MDR substrates suggests that these were the substances that fueled the evolution of MDR pumps. Two factors apparently favored this particular class of molecules for the role of original MDR substrates--need and opportunity. Unlike other substances, amphipathic cations accumulate in the cell driven by the membrane potential, which makes cations potentially the most dangerous toxins. At the same time, amphipathic cations are highly hydrated and do not permeate the membrane as readily as neutral compounds, making it feasible to design a defense based on an efflux pump. The paucity of known cationic (non-basic) antimicrobials might be a result of using MDR-expressing microbial cells for antibiotic discovery. Plant amphipathic cations, the berberine alkaloids, are good MDR substrates. The Berberis plants produce 5'-methoxyhydnocarpin-D, an MDR inhibitor that potentiates the action of berberine. It is suggested that the further evolution of MDR pumps was determined largely by the barrier function of the membrane they reside in. Thus Gram negative bacteria have an outer membrane barrier that slows the penetration of virtually all amphipathic molecules, and transenvelope MDRs of the RND and EmrAB-type extrude their substrates across this barrier. A low permeability of the cytoplasmic membrane of yeast similarly allows for the operation of broad-specificity ABC and MF MDRs. The presence of MDR sensors that regulate the expression of some MDR pumps strongly suggests that defense against external toxins is the function of these MDRs. The BmrR transcriptional activator of the MerR family induces expression of the Bmr pump in B. subtilis and is a sensor specifically designed to recognize amphipathic cations. Similarly, the OacR repressor binds chemically unrelated cations, which leads to the expression of the QacA pump in S. aureus. In E. coli, the EmrR sensor of the MarR repressor family binds unrelated neutral molecules, allowing for expression of the transenvelope EmrAB pump.

Anti-Bacterial Agents↗
PubMed2005

Directed evolution of copy number of a broad host range plasmid for metabolic engineering.

Random mutagenesis and directed evolution has been successfully used to improve desired properties of enzymes for biocatalysis and metabolic engineering. Here we employ the method to increase copy number of a pBBR-based broad host range plasmid, which can be used to express desired enzymes in a variety of microbial hosts. Localized random mutagenesis was performed in the replication control region of a pBBR-derived plasmid containing a beta-carotene reporter. Mutant plasmids were isolated that showed increased beta-carotene production. Real-time PCR analysis confirmed that the copy number of the mutant plasmids increased 3-7 fold. Sequence of the 10 mutant plasmids indicated that each plasmid contained single or multiple mutations in the rep gene or the flanking regions. Single amino acid change of serine to leucine at codon 100 of the replication protein and single nucleotide change of C to T at 46 bp upstream of the rep gene caused the increase of plasmid copy number. The utility of the mutant plasmids for metabolic engineering were further demonstrated by increased beta-carotene production, when an isoprenoid pathway gene (dxs) was co-expressed on a compatible plasmid. The mutant plasmids were tested in Agrobacterium tumefaciens. Increase of plasmid copy number and beta-carotene production was also observed in the non-Escherichia coli host.

Directed Molecular Evolution↗
PubMed2004

The ORFanage: an ORFan database.

As each newly sequenced genome contains a significant number of protein-coding ORFs that are species-, family- or lineage-specific, many interesting questions arise about the evolution and role of these ORFs and of the genomes they are part of. We refer to these poorly conserved ORFs as singleton or paralogous ORFans if they are unique to one genome, or as orthologous ORFans if they appear only in a family of closely related organisms and have no homolog in other genomes. In order to study and classify ORFans we have constructed the ORFanage, an ORFan database. This database consists of the predicted ORFs in fully sequenced microbial genomes, and enables searching for the three types of ORFans in any subset of the genomes chosen by the user. The ORFanage could help in choosing interesting targets for further genomic and evolutionary studies. The ORFanage is accessible via http://www.bioinformatics.buffalo. edu/ORFanage.

Computational Biology↗
PubMed1975

Soil fungistasis: role of the microbial nutrient sink and of fungistatic substances in two soils.

Sensitivity of conidia of Cochliobolus victoriae to fungistasis decreased markedly following incubation on moist sand for at least 1 h. Germination was greater on Conover loam or on sand being leached with water than on an alkaline clay loam soil known to produce a volatile fungistatic substance. Evolution of 14CO2 began within 3 min after [14C]glucose was applied to the soils; the rate of 14CO2 evolution was faster with Conover loam. Germination of Thielaviopsis basicola conidia per unit of glucose remaining in agar discs initially containing 0-1% glucose, was lower for discs incubated on the clay loam soil than on Conover loam, and was greatest on a bed of sand undergoing aqueous leaching. Germination of ascospores of Neurospora tetrasperma and conidia of C. victoriae was suppressed on discs of washed, Purified Agar or polyacrylamide gel incubated on or over the clay loam soil, but no suppression resulted when discs were incubated on Conover loam. Extensive aeration of either soil did not remove its fungistatic effect. Fungistasis in Conover loam appears to be caused primarily by nutrient deprivation, whereas volatile fungistatic substances may play a major role in the clay loam soil.

Antifungal Agents↗
PubMed1976

Microbial degradation of lignocellulose: the lignin component.

A new procedure was developed for the study of lignin biodegradation by pure or mixed cultures of microorganisms. Natural lignocelluloses were prepared containing C in primarily their lignin components by feeding plants l-[U-C]phenylalanine through their cut stems. Lignin degradation was observed in numerous soils by monitoring evolution of CO(2) from [C]lignin-labeled oak (Quercus albus), maple (Acer rubrum), and cattail (Typha latifola). An organism (Thermonospora fusca ATCC 27730) that is known to degrade cellulose but not lignin was shown to grow on lignocellulose in the presence of [C]lignocelluloses without evolution of CO(2). A known lignin degrader (a white-rot fungus, Polyporus versicolor) was shown to readily evolve CO(2) from damp C-labeled cattail and C-labeled maple.

Journal Article↗
PubMed1996

[Microbiological parameters of clinical interest in pulmonary infection in cystic fibrosis].

BACKGROUND: A 5-year study in patients with cystic fibrosis was carried out in order to gain a better understanding of the microbiological factors influencing clinical status and evolution. METHODS: Fifty-two patients were evaluated (mean age 16.6 years, range 0-36) during a 5 years-period (July 1988- July 1992). The clinical score of Shwachman and pulmonary function were evaluated at the beginning and at the end of the study period. Quantitative bacterial cultures were performed every 3 weeks recording the different colonical morphotypes of Pseudomonas aeruginosa. RESULTS: Beside the expected results regarding the prevalence of P. aeruginosa (80.7%) and Staphylococcus aureus (65.5%), a low rate of chronic infection with Burkholderia cepacia (1.9%) and high with S. maltophilia (9.6%) was found and it is worth noting the presence of Salmonella spp. in 3 patients. Microbial colonization followed the classical age-related sequence with 53.8% of patients older than 10 colonized with mucoid strains of P. aeruginosa. Colonization with mucoid Pseudomonas, increase in sputum bacterial counts and high diversity in colonizing morphotypes were parameters related with reduced clinical scores. CONCLUSIONS: The sequential study of the bacterial colonization in cystic fibrosis is important to follow the prognosis and evolution of the disease, and therefore, to choose the most effective therapy.

Adolescent↗
PubMed2001

Bacteriophytochromes are photochromic histidine kinases using a biliverdin chromophore.

Phytochromes comprise a principal family of red/far-red light sensors in plants. Although phytochromes were thought originally to be confined to photosynthetic organisms, we have recently detected phytochrome-like proteins in two heterotrophic eubacteria, Deinococcus radiodurans and Pseudomonas aeruginosa. Here we show that these form part of a widespread family of bacteriophytochromes (BphPs) with homology to two-component sensor histidine kinases. Whereas plant phytochromes use phytochromobilin as the chromophore, BphPs assemble with biliverdin, an immediate breakdown product of haem, to generate photochromic kinases that are modulated by red and far-red light. In some cases, a unique haem oxygenase responsible for the synthesis of biliverdin is part of the BphP operon. Co-expression of this oxygenase with a BphP apoprotein and a haem source is sufficient to assemble holo-BphP in vivo. Both their presence in many diverse bacteria and their simplified assembly with biliverdin suggest that BphPs are the progenitors of phytochrome-type photoreceptors.

Amino Acid Sequence↗