Development of a genetic system for Geobacter sulfurreducens.

Members of the genus Geobacter are the dominant metal-reducing microorganisms in a variety of anaerobic subsurface environments and have been shown to be involved in the bioremediation of both organic and metal contaminants. To facilitate the study of the physiology of these organisms, a genetic system was developed for Geobacter sulfurreducens. The antibiotic sensitivity of this organism was characterized, and optimal conditions for plating it at high efficiency were established. A protocol for the introduction of foreign DNA into G. sulfurreducens by electroporation was also developed. Two classes of broad-host-range vectors, IncQ and pBBR1, were found to be capable of replication in G. sulfurreducens. In particular, the IncQ plasmid pCD342 was found to be a suitable expression vector for this organism. When the information and novel methods described above were utilized, the nifD gene of G. sulfurreducens was disrupted by the single-step gene replacement method. Insertional mutagenesis of this key gene in the nitrogen fixation pathway impaired the ability of G. sulfurreducens to grow in medium lacking a source of fixed nitrogen. Expression of the nifD gene in trans complemented this phenotype. This paper constitutes the first report of genetic manipulation of a member of the Geobacter genus.     

Application of phenotypic microarrays to environmental microbiology

Environmental organisms are extremely diverse and only a small fraction has been successfully cultured in the laboratory. Culture in micro wells provides a method for rapid screening of a wide variety of growth conditions and commercially available plates contain a large number of substrates, nutrient sources, and inhibitors, which can provide an assessment of the phenotype of an organism. This review describes applications of phenotype arrays to anaerobic and thermophilic microorganisms, use of the plates in stress response studies, in development of culture media for newly discovered strains, and for assessment of phenotype of environmental communities. Also discussed are considerations and challenges in data interpretation and visualization, including data normalization, statistics, and curve fitting.     

The adaptive genome of Desulfovibrio vulgaris Hildenborough

Peculiar attributes revealed by sequencing the genome of Desulfovibrio vulgaris Hildenborough are analyzed, particularly in relation to the presence of a phosphotransferase system (PTS). The PTS is a typical bacterial carbohydrate transport system functioning via group translocation. Novel avenues for investigations are proposed emphasizing the metabolic diversity of D. vulgaris Hildenborough, especially the likely utilization of mannose-type sugars. Comparative analysis with PTS from other Gram-negative and Gram-positive bacteria indicates regulatory functions for the PTS of D. vulgaris Hildenborough, including catabolite repression and inducer exclusion. Chemotaxis towards PTS substrates is considered. Evidence suggests that this organism may not be a strict anaerobic sulfate reducer typical of the ocean, but a versatile organism capable of bidirectional transmigration and adaptation to both water and terrestrial environments.     

Five-gene cluster in Clostridium thermoaceticum consisting of two divergent operons encoding rubredoxin oxidoreductase- rubredoxin and rubrerythrin-type A flavoprotein- high-molecular-weight rubredoxin

A five-gene cluster encoding four nonheme iron proteins and a flavoprotein from the thermophilic anaerobic bacterium Clostridium thermoaceticum (Moorella thermoacetica) was cloned and sequenced. Based on analysis of deduced amino acid sequences, the genes were identified as rub (rubredoxin), rbo (rubredoxin oxidoreductase), rbr (rubrerythrin), fprA (type A flavoprotein), and a gene referred to as hrb (high-molecular-weight rubredoxin). Northern blot analysis demonstrated that the five-gene cluster is organized as two subclusters, consisting of two divergently transcribed operons, rbr-fprA-hrb and rbo-rub. The rbr, fprA, and rub genes were expressed in Escherichia coli, and their encoded recombinant proteins were purified. The molecular masses, UV-visible absorption spectra, and cofactor contents of the recombinant rubrerythrin, rubredoxin, and type A flavoprotein were similar to those of respective homologs from other microorganisms. Antibodies raised against Desulfovibrio vulgaris Rbr reacted with both native and recombinant Rbr from C. thermoaceticum, indicating that this protein was expressed in the native organism. Since Rbr and Rbo have been recently implicated in oxidative stress protection in several anaerobic bacteria and archaea, we suggest a similar function of these proteins in oxygen tolerance of C. thermoaceticum.     

Degradation of BTEX by anaerobic bacteria: physiology and application

Pollution of the environment with aromatic hydrocarbons, such as benzene, toluene, ethylbenzene and xylene (so-called BTEX) is often observed. The cleanup of these toxic compounds has gained much attention in the last decades. In situ bioremediation of aromatic hydrocarbons contaminated soils and groundwater by naturally occurring microorganisms or microorganisms that are introduced is possible. Anaerobic bioremediation is an attractive technology as these compounds are often present in the anoxic zones of the environment. The bottleneck in the application of anaerobic techniques is the lack of knowledge about the anaerobic biodegradation of benzene and the bacteria involved in anaerobic benzene degradation. Here, we review the existing knowledge on the degradation of benzene and other aromatic hydrocarbons by anaerobic bacteria, in particular the physiology and application, including results on the (per)chlorate stimulated degradation of these compounds, which is an interesting new alternative option for bioremediation.     

The ecological determinacy of the intraspecies diversity of pathogenic bacteria

The review deals with some theoretical and applied problems of the intraspecific diversity of pathogenic microorganisms. Special attention is given to the role of ecological factors in the formation of the phenotypic polymorphism of bacteria. The possibility of using the methods of mathematical analysis for the evaluation of the influence of the environment (ecotopes) on individual phenotypic characteristics of bacteria and their complex (bioprofile) is shown. The proposition on the bioprofile of microorganisms as a criterion of their ecological (ecotopic) marking is substantiated. The examples of using the data of the ecological marking of pathogenic bacteria in clinical epidemiology and ecological practice are presented.     

Identification of crude-oil components and microorganisms that cause souring under anaerobic conditions

Oil souring has important implications with respect to energy resources. Understanding the physiology of the microorganisms that play a role and the biological mechanisms are both important for the maintenance of infrastructure and mitigation of corrosion processes. The objective of this study was to identify crude-oil components and microorganisms in oil-field water that contribute to crude-oil souring. To identify the crude-oil components and microorganisms that are responsible for anaerobic souring in oil reservoirs, biological conversion of crude-oil components under anaerobic conditions was investigated. Microorganisms in oil field water in Akita, Japan degraded alkanes and aromatics to volatile fatty acids (VFAs) under anaerobic conditions, and fermenting bacteria such as Fusibacter sp. were involved in VFA production. Aromatics such as toluene and ethylbenzene were degraded by sulfate-reducing bacteria (Desulfotignum sp.) via the fumarate-addition pathway and not only degradation of VFA but also degradation of aromatics by sulfate-reducing bacteria was the cause of souring. Naphthenic acid and 2,4-xylenol were not converted.     

The aerobic and anaerobic microbiology of pustular acne lesions

Specimens from 32 pustular acne lesions that were inoculated on media supportive for the growth of aerobic and anaerobic bacteria showed bacterial growth. Only aerobic or facultative bacteria were recovered in 15 (47%) specimens, only anaerobic bacteria in 11 (34%) specimens, and mixed aerobic and anaerobic bacteria in 6 (18%) specimens. A total of 57 isolates, 31 anaerobes (1.0 per specimen) and 26 aerobes (0.8 per specimen) were recovered. The predominant isolates were Staphylococcus sp. (19 isolates), Peptostreptococcus sp. (15), and Propionibacterium sp. (10). Twelve (37.5%) of the comedones yielded only one organism. This retrospective study highlighted the polymicrobial nature of over two-thirds of culture positive pustular acne lesions and suggests the potential for pathogenic role of aerobic and anaerobic organisms other than P. acnes and Staphylococcus sp. in acne vulgaris.     

Using time series analysis to characterize evolutionary and plastic responses to environmental change: a case study of a shift toward earlier migration date in sockeye salmon

Environmental change can shift the phenotype of an organism through either evolutionary or nongenetic processes. Despite abundant evidence of phenotypic change in response to recent climate change, we typically lack sufficient genetic data to identify the role of evolution. We present a method of using phenotypic data to characterize the hypothesized role of natural selection and environmentally driven phenotypic shifts (plasticity). We modeled historical selection and environmental predictors of interannual variation in mean population phenotype using a multivariate state-space model framework. Through model comparisons, we assessed the extent to which an estimated selection differential explained observed variation better than environmental factors alone. We applied the method to a 60-year trend toward earlier migration in Columbia River sockeye salmon Oncorhynchus nerka, producing estimates of annual selection differentials, average realized heritability, and relative cumulative effects of selection and plasticity. We found that an evolutionary response to thermal selection was capable of explaining up to two-thirds of the phenotypic trend. Adaptive plastic responses to June river flow explain most of the remainder. This method is applicable to other populations with time series data if selection differentials are available or can be reconstructed. This method thus augments our toolbox for predicting responses to environmental change.     

Anaerobic microflora of the female reproductive tract

The microflora of the female reproductive tract is very diverse and plays an important role in both normal and pathological states. The data on the mechanisms of colonization resistance which involve the vaginal microbios (the production of H2O2, organic acids, bacteriocin-like substances, competition for adhesion sites) are presented. The data on the role of individual antagonistically active substances of anaerobic bacteria in suppressing gonococci, fungi, microorganisms, associated with bacterial vaginosis, etc. are given. The leading role of anaerobic microorganisms in the appearance of microecological disturbances, including bacterial vaginosis, is emphasized. The role of the pathogenic properties of anaerobic bacteria for the development of different pathological processes, such as premature birth, postnatal and postoperative purulent septic diseases, inflammation of pelvic organs, cancer of the neck of uterus, is discussed.     

Monitoring the enrichment and isolation of sulfate-reducing bacteria by using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences.

A fluorescently labeled version of a population-specific oligonucleotide hybridization probe was used to monitor the enrichment and isolation of a sulfate-reducing bacterium from a multispecies anaerobic bioreactor. The organism was originally identified as a molecular isolate that was phylogenetically related to Desulfovibrio vulgaris by amplification and sequencing of part of its 16S rRNA sequence. The sequence, in turn, was used to design a population-specific probe. The anaerobic medium used for the organism's enrichment and isolation was based on the physiological properties of the its closest relatives as identified by sequence comparisons. Of 30 isolates examined, only 3 hybridized with the probe. Nearly complete 16S rRNA sequences determined for each of these three isolates (i) had no mismatches with the probe target site, (ii) were identical to the amplified partial sequence of about 500 nucleotides and to one another in all other positions, and (iii) were 93.9% similar to that of D. vulgaris. In addition, one isolate chosen for further study (strain PT-2) had a substrate specificity comparable to that of D. vulgaris. These results confirmed that polymerase chain reaction amplification of 16S rRNA sequences from environmental samples can be accurate and can also provide phylogenetic information from which aspects of a population's physiology can be inferred.     

Molecular microbiology of gut bacteria: genetic diversity and community structure analysis

Recently developed molecular biology approaches make possible the detailed genetic, taxonomic and ecological examination of microorganisms from various habitats. Animal gut represents one of the most complex microbial ecosystems with a large degree of microbial biodiversity present. Bacteria inhabiting the gut usually play important roles in metabolic transformations of substrates and sometimes, e.g. in ruminants, they make the basis for an obligate symbiosis with the host. Here we discuss molecular microbiology as a strategy for examination of gut bacteria, concentrating on a typical and in such environment dominant group of strictly anaerobic Gram-negative bacteria from the phylogenetic group Cytophaga/Flexibacter/Bacteroides. The bacteria from the genus Prevotella are the most abundant Gram-negative bacteria in the rumen and form a distinctive phylogenetic cluster, clearly separated from prevotellas isolated from other ecological niches. They may represent a good choice for a model organism in genetic manipulation experiments and for studies of gene transfer mechanisms taking place in the gut. The molecular tools for detection and monitoring of ruminal prevotellas are discussed.     

How Vibrio cholerae survive during starvation

Vibrio cholerae, a Gram-negative, motile, aquatic bacterium, is the causal agent of the diarrheal disease cholera. Cholera is a serious epidemic disease that has killed millions of people and continues to be a major health problem world-wide. The hypothesis that V. cholerae occupies an ecological niche in the estuarine environment requires that this organism is able to survive the dynamics of physiochemical stresses, including nutrient starvation. As a result of these stresses, bacteria in nature often exist in non-growth or very slow growth states with a low metabolic activity. Because microorganisms have little ability to control their environment, environmental changes have led to changes in cell function and structure. Such cellular responses can originate in one of two ways: by changes in genetic constitution or by phenotypic adaptation. In this review, we will focus on the phenotypic responses of V. cholerae of a given genotype to starvation stress.     

Recent advances in domesticating non-model microorganisms

Non-model microorganisms have been increasingly explored as microbial cell factories for production of chemicals, fuels, and materials owing to their unique physiology and metabolic capabilities. However, these microorganisms often lack facile genetic tools for strain development, which hinders their adoption as production hosts. In this review, we describe recent advances in domestication of non-model microorganisms, including bacteria, actinobacteria, cyanobacteria, yeast, and fungi, with a focus on the development of genetic tools. In addition, we highlight some successful applications of non-model microorganisms as microbial cell factories.     

Biodegradation of Phenol: Mechanisms and Applications

Phenol, or hydroxybenzene, is both a synthetically and naturally produced aromatic compound. Microorganisms capable of degrading phenol are common and include both aerobes and anaerobes. Many aerobic phenol-degrading microorganisms have been isolated and the pathways for the aerobic degradation of phenol are now firmly established. The first steps include oxygenation of phenol by phenol hydroxylase enzymes to form catechol, followed by ring cleavage adjacent to or in between the two hydroxyl groups of catechol. Phenol hydroxylases ranging from simple flavoprotein monooxygenases to multicomponent hydroxylases, as well as the genes coding for these enzymes, have been described for a number of aerobic phenol-degrading microorganisms. Phenol can also be degraded in the absence of oxygen. Our knowledge of this process is less advanced than that of the aerobic process, and only a few anaerobic phenol-degrading bacteria have been isolated to date. Convincing evidence from both pure culture studies with the denitrifying organism Thauera aromatica K172 and with two Clostridium species, as well as from mixed culture studies, indicates that the first step in anaerobic phenol degradation is carboxylation in the para-position to form 4-hydroxybenzoate. Following para-carboxylation, thioesterification of 4-hydroxybenzoate to co-enzyme A allows subsequent ring reduction, hydration, and fission. Para-carboxylation appears to be involved in the anaerobic degradation of a number of aromatic compounds. Numerous practical applications exist for microbial phenol degradation. These include the exploitation of indigenous anaerobic phenol-degrading bacteria in the in situ bioremediation of creosote-contaminated subsurface environments, and the use of phenol as a co-substrate for indigenous aerobic phenol-degrading bacteria to enhance in situ biodegradation of chlorinated solvents.     

Fowl cholera outbreak in domestic poultry and epidemiological properties of Pasteurella multocida isolate

Symptoms of fowl cholera including orofacial edema, swollen and edematous wattles and combs, and severe respiratory disorders were detected in domestic poultry in two broiler breeder farms: one located in Gyeong-gi Province (October, 2000) and the other in Chung-cheong-nam Province (March, 2001). Gram-negative, bipolar staining bacillus was easily found in a direct smear. The biochemical properties of isolates were examined using a standard diagnosis method, proving that they were 99.7% similar to the Pasteurella multocida (P. multocida: PM), a pathogenic and causative agent of fowl cholera (FC). As a result, an FC outbreak in domestic fowls was confirmed for the first time in Korea since 1942. Because FC was detected in broiler breeder farms for the first time in 59 years at the same time as an FC outbreak was confirmed in wild birds (October, 2000), our concern was focused on whether the PM strains that originated in wild birds were transmitted into poultry farms. The possibility was tracked down by comparing phenotypic and genetic properties between the two types of PM strains. PM strains of chicken origin showed prominent differences from the PM strains of wild bird origin in both phenotypic and genetic properties. An examination of the origin of the wild bird bacteria was conducted, but no evidence has been identified that PM strains from the wild bird were introduced into domestic poultry farms.     

Environmental and genetic cues in the evolution of phenotypic polymorphism

Phenotypic polymorphism is a consequence of developmental plasticity, in which the trajectories of developing organisms diverge under the influence of cues. Environmental and genetic phenotype determination are the two main categories of polymorphic development. Even though both may evolve as a response to varied environments, they are traditionally regarded as fundamentally distinct phenomena. They can however be joined into a single framework that emphasizes the parallel roles of environmental and genetic cues in phenotype determination. First, from the point of view of immediate causation, it is common that phenotypic variants can be induced either by environmental or by allelic variation, and this is referred to as gene-environment interchangeability. Second, from the point of view of adaptation, genetic cues in the form of allelic variation at polymorphic loci can play similar roles as environmental cues in providing information to the developmental system about coming selective conditions. Both types of cues can help a developing organism to fit its phenotype to selective circumstances. This perspective of information in environmental and genetic cues can produce testable hypotheses about phenotype determination, and can thus increase our understanding of the evolution of phenotypic polymorphism.