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PubMed1998

Selected animal models: vaginal candidosis, Pneumocystis pneumonia, dermatophytosis and trichosporosis.

A clear understanding of the pathogenesis of fungal disease remains elusive. While technological advances in molecular biology and microbial genetics have provided scientists with major new insights into both microbial virulence factors as well as host susceptibility to infection, there is currently no substitute for animal models in elucidating microbe-host interactions. Animal models are also essential for the evaluation of new antimicrobial agents, including studies of efficacy, adverse reactions and pharmacokinetics. The single most important advance in animal models in the last decade, has been the availability of genetically unique strains of animals as alternative to animals treated with immunosuppressive drugs for use in studies on microbial virulence and host defence mechanisms. These unique strains of test animals also enhance our understanding of the modes of action of antifungal drugs and their metabolism. Some of these advances will be discussed in this symposium.

Animals↗
PubMed1993

Evolution of prokaryotic genomes.

Molecular genetics, which has its roots mainly in the development of microbial genetics in the middle of this century, not only greatly facilitates investigations of essential cellular functions, but also offers a means to better understand evolutionary progress. Spontaneous mutagenesis, the driving force of biological evolution, depends on a multitude of mechanistically distinct processes, many of which are already quite well understood. Often, enzymes act as variation generators, and natural gene vectors help to spread functional domains, entire genes and groups of genes across natural isolation barriers. In this overview, particular attention is given to comparing three selected natural strategies for the generation of genetic diversity: nucleotide substitution, DNA rearrangements, and gene acquisition. All of these mechanisms, as well as many others, appear to fulfill their specific roles in microbial evolution. Rather than being the result of an accumulation of errors, biological evolution may depend on a multitude of specific biological functions, as well as on a certain degree of intrinsic structural flexibility of biological molecules.

Biological Evolution↗
PubMed2004

Acinetobacter sp. ADP1: an ideal model organism for genetic analysis and genome engineering.

Acinetobacter sp. strain ADP1 is a naturally transformable gram-negative bacterium with simple culture requirements, a prototrophic metabolism and a compact genome of 3.7 Mb which has recently been sequenced. Wild-type ADP1 can be genetically manipulated by the direct addition of linear DNA constructs to log-phase cultures. This makes it an ideal organism for the automation of complex strain construction. Here, we demonstrate the flexibility and versatility of ADP1 as a genetic model through the construction of a broad variety of mutants. These include marked and unmarked insertions and deletions, complementary replacements, chromosomal expression tags and complex combinations thereof. In the process of these constructions, we demonstrate that ADP1 can effectively express a wide variety of foreign genes including antibiotic resistance cassettes, essential metabolic genes, negatively selectable catabolic genes and even intact operons from highly divergent bacteria. All of the described mutations were achieved by the same process of splicing PCR, direct transformation of growing cultures and plating on selective media. The simplicity of these tools make genetic analysis and engineering with Acinetobacter ADP1 accessible to laboratories with minimal microbial genetics expertise and very little equipment. They are also compatible with complete automation of genetic analysis and engineering protocols.

Acinetobacter↗
PubMed2006

Microbioreactor arrays with integrated mixers and fluid injectors for high-throughput experimentation with pH and dissolved oxygen control.

We have developed an integrated array of microbioreactors, with 100 microL working volume, comprising a peristaltic oxygenating mixer and microfluidic injectors. These integrated devices were fabricated in a single chip and can provide a high oxygen transfer rate (k(L)a approximately 0.1 s(-1)) without introducing bubbles, and closed loop control over dissolved oxygen and pH (+/-0.1). The system was capable of supporting eight simultaneous Escherichia coli fermentations to cell densities greater than 13 g-dcw L(-1) (1 cm OD(650 nm) > 40). This cell density was comparable to that achieved in a 4 litre reference fermentation, conducted with the same strain, in a bench scale stirred tank bioreactor and is more than four times higher than cell densities previously achieved in microbioreactors. Bubble free oxygenation permitted near real time optical density measurements which could be used to observe subtle changes in the growth rate and infer changes in the state of microbial genetic networks. Our system provides a platform for the study of the interaction of microbial populations with different environmental conditions, which has applications in basic science and industrial bioprocess development. We leverage the advantages of microfluidic integration to deliver a disposable, parallel bioreactor in a single chip, rather than robotically multiplexing independent bioreactors, which opens a new avenue for scaling small scale bioreactor arrays with the capabilities of bench scale stirred tank reactors.

Ammonium Hydroxide↗
PubMed1999

Food microbiology: the challenges for the future.

Food microbiology has become a mature science in the twentieth century and has made great advances. While recognising these achievements, it is also necessary to consider how the science may need to change. This paper addresses this by reference to three areas. These are possible changes in foodborne diseases of concern and the impact of molecular and genetic techniques on our current methodology. The recognition of the role of food and associated microbial contaminants in chronic diseases could become a major concern. New developments in our understanding of microbial genetics could affect our concepts of bacterial taxonomy. The current methodologies we use, based upon genotypically identical populations, may need to be addressed. If the trends indicated here are realised, they indicate a major challenge and opportunity for the food microbiologist.

Food Microbiology↗
PubMed1997

A genetic system to identify DNA polymerase beta mutator mutants.

DNA polymerase beta (pol beta) is a 39-kDa protein that functions in DNA repair processes in mammalian cells. As a first step toward understanding mechanisms of polymerase fidelity, we developed a genetic method to identify mammalian pol beta mutator mutants. This screen takes advantage of a microbial genetics assay and the ability of rat pol beta to substitute for Escherichia coli DNA polymerase I in DNA replication in vivo. Using this screen, we identified 13 candidate pol beta mutator mutants. Three of the candidate mutator mutants were further characterized in vivo and shown to confer an increased spontaneous mutation frequency over that of wild-type pol beta to our bacterial strain. Purification and subsequent analysis of one of our putative mutator proteins, the pol beta-14 protein, showed that it possesses intrinsic mutator activity in four different assays that measure the fidelity of DNA synthesis. Therefore, residue 265, which is altered in pol beta-14 and another of our mutant proteins, pol beta-166, is probably critical for accurate DNA synthesis by pol beta. Thus, our genetic method of screening for pol beta mutator mutants is useful in identifying active mammalian DNA polymerase mutants that encode enzymes that catalyze DNA synthesis with altered fidelity compared with the wild-type pol beta enzyme.

Animals↗
PubMedDate not supplied

Molecular Koch's postulates applied to microbial pathogenicity.

Microbial genetics and molecular cloning now permit us to routinely isolate specific genes from a variety of microbial pathogens. Obviously not all genes from pathogenic microorganisms play a role in pathogenicity or virulence. Just as Koch's postulates were formulated to identify the causal relationship between an organism and a specific disease, the notion is presented here that a form of molecular Koch's postulates is needed when examining the potential role of genes and their products in the pathogenesis of infection and disease.

Animals↗
PubMed2005

Gene products with evolutionary functions.

It is often tacitly assumed that all gene products serve the needs of life functions of the individual carrying the genome. However, a close look at the formation of genetic variations, which are the drivers of biological evolution, reveals a different view. While a majority of the products of genes, such as housekeeping genes and genes essential for each individual, when exposed to particular life conditions respond to the definition given above, other gene products clearly carry out evolutionary functions at the level of populations. Products of these evolution genes act as generators of genetic variations and/or as modulators of the frequency of genetic variation. This is most readily seen with bacterial populations. Many different mechanisms contribute to the occasional, overall formation of genetic variations. These mechanisms can be grouped into three mechanistically and qualitatively different strategies of generating genetic variations. In addition to the activities of evolution genes, specific properties of matter such as tautomery also contribute to the formation of genetic variations. The views that nature cares actively for biological evolution are documented by evidence taken mainly from microbial genetics. Essential elements of the theory of molecular evolution are discussed, as well as the relevance of this theory for higher organisms and its impact on our worldview.

Animals↗
PubMed1994

Pollutant effects on the microbial ecosystem.

Genetic diversity of a microbial community will inevitably be affected by environmental stress. However, our understanding of the implications of these effects is limited. Genetic exchange between natural microbial communities appears to be a common phenomenon, mediated by a number of microbial processes (conjugation, transformation, and transduction). These mechanisms of change are presumably adaptations to natural environmental perturbation, e.g., the low levels of antibiotics produced by other organisms. However, anthropogenic influences on the environment may be accelerating genetic change within microbiologic ecosystems, beyond these natural adaptation rates. This article highlights some of the perceived risks to ecosystem health and research questions that need to be addressed.

Colony Count, Microbial↗
PubMed2002

Phylogenetic diversity of marine cyanophage isolates and natural virus communities as revealed by sequences of viral capsid assembly protein gene g20.

In order to characterize the genetic diversity and phylogenetic affiliations of marine cyanophage isolates and natural cyanophage assemblages, oligonucleotide primers CPS1 and CPS8 were designed to specifically amplify ca. 592-bp fragments of the gene for viral capsid assembly protein g20. Phylogenetic analysis of isolated cyanophages revealed that the marine cyanophages were highly diverse yet more closely related to each other than to enteric coliphage T4. Genetically related marine cyanophage isolates were widely distributed without significant geographic segregation (i.e., no correlation between genetic variation and geographic distance). Cloning and sequencing analysis of six natural virus concentrates from estuarine and oligotrophic offshore environments revealed nine phylogenetic groups in a total of 114 different g20 homologs, with up to six clusters and 29 genotypes encountered in a single sample. The composition and structure of natural cyanophage communities in the estuary and open-ocean samples were different from each other, with unique phylogenetic clusters found for each environment. Changes in clonal diversity were also observed from the surface waters to the deep chlorophyll maximum layer in the open ocean. Only three clusters contained known cyanophage isolates, while the identities of the other six clusters remain unknown. Whether or not these unidentified groups are composed of bacteriophages that infect different Synechococcus groups or other closely related cyanobacteria remains to be determined. The high genetic diversity of marine cyanophage assemblages revealed by the g20 sequences suggests that marine viruses can potentially play important roles in regulating microbial genetic diversity.

Bacteriophages↗
PubMed1972

Intergenotic transformation of the Bacillus subtilis genospecies.

A multiple auxotrophic derivative of Bacillus subtilis 168 (strain BR151 carrying lys-3, trpC2, metB10) was transformed with deoxyribonucleic acid (DNA) isolated from B. subtilis 168, Bacillus amyloliquefaciens H, B. subtilis HSR, Bacillus pumilus, and Bacillus licheniformis. Transformation with heterologous DNA occurred at a very low frequency for the three auxotrophic markers. Heterologous transformation to rifampin resistance was 100 to 1,000 times more efficient than transformation to prototrophy. Transformants from the various heterologous exchanges were used to prepare donor DNA. The fragment of integrated DNA from the heterologous (foreign) species, termed the "intergenote," was capable of transforming BR151 with an efficiency almost equal to that of homologous DNA. When BR151 DNA contained the Rfm(R) (rifampin resistance) intergenote from B. amyloliquefaciens H, the frequency of transformation was frequently greater than that of the homologous DNA. Accompanying this increased efficiency was a marked change in the physiology of the cells. The growth rate of the transformants carrying this intergenote was approximately one-half that of either parental strain. Thus, in a prokaryotic transformation system, adverse side effects can occur after incorporation of a segment of foreign DNA.

Bacillus subtilis↗