Explore the vast range of titles available.

Transcription factors regulate gene expression through their binding to DNA. In a living Escherichia coli cell, we directly observed specific binding of a lac repressor, labeled with a fluorescent protein, to a chromosomal lac operator. Using single-molecule detection techniques, we measured the kinetics of binding and dissociation of the repressor in response to metabolic signals. Furthermore, we characterized the nonspecific binding to DNA, one-dimensional (1D) diffusion along DNA segments, and 3D translocation among segments through cytoplasm at the single-molecule level. In searching for the operator, a lac repressor spends ~90% of time nonspecifically bound to and diffusing along DNA with a residence time of <5 milliseconds. The methods and findings can be generalized to other nucleic acid binding proteins.

Gene inventory and metagenomic techniques have allowed rapid exploration of bacterial diversity and the potential physiologies present within microbial communities. However, it remains nontrivial to discover the identities of environmental bacteria carrying two or more genes of interest. We have used microfluidic digital polymerase chain reaction (PCR) to amplify and analyze multiple, different genes obtained from single bacterial cells harvested from nature. A gene encoding a key enzyme involved in the mutualistic symbiosis occurring between termites and their gut microbiota was used as an experimental hook to discover the previously unknown ribosomal RNA-based species identity of several symbionts. The ability to systematically identify bacteria carrying a particular gene and to link any two or more genes of interest to single species residing in complex ecosystems opens up new opportunities for research on the environment.

This article elaborates on possible future directions for microbial viability assessment using nucleic acid-modifying compounds in combination with DNA- (and potentially RNA-) amplification technologies. Bacteria were traditionally considered viable when they could be cultured, whereas today's viability concept is based on the presence of some form of metabolic activity, responsiveness, RNA transcripts that tend to degrade rapidly after cell death, or of an intact membrane. The latter criterion was the focus of recent approaches to limit detection to intact cells using ethidium monoazide or propidium monoazide. Membrane integrity must, however, be considered as a very conservative criterion for microbial viability. The new concept presented here aims at limiting nucleic acid-based detection to cells with an active metabolism, which might be a more appropriate viability criterion. To selectively detect only cells with metabolic and respiratory activity (while excluding inactive dead cells from detection), we suggest the use of 'activity-labile compounds'. In addition to their potential usefulness for viability assessment, these new compounds could also be beneficial for selectively amplifying nucleic acids of cells that have metabolic activities of interest. This preferential detection of microorganisms with certain metabolic capabilities is referred to as 'molecular enrichment' in distinction to 'growth enrichment'.

Since the invention of the Petri dish, there have been continuous efforts to improve efficiency in microbial cultivation. These efforts were devoted to the attainment for diverse growth conditions, simulation of in situ conditions and achievement of high-throughput rates. As a result, prokaryotes catalysing novel redox reactions as well as representatives of abundant, but not-yet cultured taxa, were isolated. Significant insights into microbial physiology have been made by studying the small number of prokaryotes already cultured. However, despite these numerous breakthroughs, microbial cultivation is still a low-throughput process. The main hindrance to cultivation is likely due to the prevailing lack of knowledge on targeted species. In this review, we focus on the limiting factors surrounding cultivation. We discuss several cultivation obstacles, including the loss of microbial cell-cell communication following species isolation. Future research directions, including the refinement of culture media, strategies based on cell-cell communication and high-throughput innovations, are reviewed. We further propose that a combination of these approaches is urgently required to promote cultivation of uncultured species, thereby dawning a new era in the field.

Natural gas guzzlers There has been an increasing interest in organisms thriving at marine gas seeps, in particular microbes that utilize methane. Surprisingly, there seems to have been comparatively little work done on the fate of the other abundant hydrocarbons in natural gases — ethane, propane and butane. Now sediments collected from hydrocarbon seep areas in the Gulf of Mexico and the Gulf of California have yielded microbial cultures that utilize propane and butane under anoxic conditions similar to those prevailing in gas reservoirs. These biochemically unusual bacteria may be responsible for the observed alteration of gases in seeps and other gas reservoirs. Ethane, propane and butane are constituents of natural gas in anoxic marine sediments. The first microbial isolate belonging to the sulphate-reducing bacteria capable of anaerobically oxidizing short-chain non-methane hydrocarbons is described. The short-chain hydrocarbons ethane, propane and butane are constituents of natural gas. They are usually assumed to be of thermochemical origin 1 , but biological formation of ethane and propane has been also observed 2 . Microbial utilization of short-chain hydrocarbons has been shown in some aerobic species 3 , 4 but not in anaerobic species of bacteria. On the other hand, anaerobic utilization of short-chain hydrocarbons would in principle be expected because various anaerobic bacteria grow with higher homologues (≥C 6 ) 5 . Indeed, chemical analyses of hydrocarbon-rich habitats with limited or no access of oxygen indicated in situ biodegradation of short-chain hydrocarbons 6 , 7 , 8 , 9 , 10 . Here we report the enrichment of sulphate-reducing bacteria (SRB) with such capacity from marine hydrocarbon seep areas. Propane or n -butane as the sole growth substrate led to sediment-free sulphate-reducing enrichment cultures growing at 12, 28 or 60 °C. With ethane, a slower enrichment with residual sediment was obtained at 12 °C. Isolation experiments resulted in a mesophilic pure culture (strain BuS5) that used only propane and n -butane (methane, isobutane, alcohols or carboxylic acids did not support growth). Complete hydrocarbon oxidation to CO 2 and the preferential oxidation of 12 C-enriched alkanes were observed with strain BuS5 and other cultures. Metabolites of propane included iso- and n -propylsuccinate, indicating a subterminal as well as an unprecedented terminal alkane activation with involvement of fumarate. According to 16S ribosomal RNA analyses, strain BuS5 affiliates with Desulfosarcina / Desulfococcus , a cluster of widespread marine SRB. An enrichment culture with propane growing at 60 °C was dominated by Desulfotomaculum -like SRB. Our results suggest that diverse SRB are able to thrive in seep areas and gas reservoirs on propane and butane, thus altering the gas composition and contributing to sulphide production.

Ruminal microorganisms hydrogenate polyunsaturated fatty acids (PUFA) present in forages and thereby restrict the availability of health-promoting PUFA in meat and milk. The aim of this study was to investigate PUFA metabolism and the influence of PUFA on members of the ruminal microflora. Eleven of 26 predominant species of ruminal bacteria metabolised linoleic acid (LA; cis-9,cis-12-18:2) substantially. The most common product was vaccenic acid (trans-11-18:1), produced by species related to Butyrivibrio fibrisolvens. α-Linolenic acid (LNA; cis-9,cis-12,cis-15-18:3) was metabolised mostly by the same species. The fish oil fatty acids, eicosapentaenoic acid (EPA; 20:5(n - 3)) and docosahexaenoic acid (DHA; 22:6(n - 3)) were not metabolised. Cellulolytic bacteria did not grow in the presence of any PUFA at 50 μg ml-¹, nor did some butyrate-producing bacteria, including the stearate producer Clostridium proteoclasticum, Butyrivibrio hungatei and Eubacterium ruminantium. Toxicity to growth was ranked EPA > DHA > LNA > LA. Cell integrity, as measured using propidium iodide, was damaged by LA in all 26 bacteria, but to different extents. Correlations between its effects on growth and apparent effects on cell integrity in different bacteria were low. Combined effects of LA and sodium lactate in E. ruminantium and C. proteoclasticum indicated that LA toxicity is linked to metabolism in butyrate-producing bacteria. PUFA also inhibited the growth of the cellulolytic ruminal fungi, with Neocallimastix frontalis producing small amounts of cis-9,trans-11-18:2 (CLA) from LA. Thus, while dietary PUFA might be useful in suppressing the numbers of biohydrogenating ruminal bacteria, particularly C. proteoclasticum, care should be taken to avoid unwanted effects in suppressing cellulolysis.

The taxonomic composition of environmental communities is an important indicator of their ecology and function. We used a set of protein-coding marker genes, extracted from large-scale environmental shotgun sequencing data, to provide a more direct, quantitative, and accurate picture of community composition than that provided by traditional ribosomal RNA-based approaches depending on the polymerase chain reaction. Mapping marker genes from four diverse environmental data sets onto a reference species phylogeny shows that certain communities evolve faster than others. The method also enables determination of preferred habitats for entire microbial clades and provides evidence that such habitat preferences are often remarkably stable over time.

Abstract Twenty-five years ago this past autumn, we published a short article entitled ‘Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity’ in Volume 9 of FEMS Microbiology Letters. Together with my Ph.D. supervisors, Eugene Rosenberg and David Gutnick, we proposed a method of measuring bacterial cell surface hydrophobicity based on bacterial adherence to hydrocarbon (‘BATH’, later known as ‘MATH’, for microbial adhesion to hydrocarbon). The method became popular soon after it was published, and the paper was, for at least the following decade, the Journal's most cited article. It became an ISI ‘citation classic’ in 1991. This minireview is a rather personal look at the development of the method and its various modifications and other scientific offspring, with the perspective of a quarter-century.

Biological nitrogen fixation, the conversion of atmospheric nitrogen to ammonia for biosynthesis, is exclusively performed by a few bacteria and archaea. Despite the essential importance of biological nitrogen fixation, it has been impossible to quantify the incorporation of nitrogen by individual bacteria or to map the fate of fixed nitrogen in host cells. In this study, with multi-isotope imaging mass spectrometry we directly imaged and measured nitrogen fixation by individual bacteria within eukaryotic host cells and demonstrated that fixed nitrogen is used for host metabolism. This approach introduces a powerful way to study microbes and global nutrient cycles.

We evaluate the substantial amount of information accumulated on bacterial diversity in a variety of environments and address several fundamental questions, focusing on aquatic systems but including other environments to provide a broader context. Bacterial diversity data were extracted from 225 16S rDNA libraries described in published reports, representing a variety of aquatic and non-aquatic environments. Libraries were predominantly composed of rare phylotypes that appeared only once or twice in the library, and the number of phylotypes observed was correlated with library size (implying that few libraries are exhaustive samples of diversity in the source community). Coverage, the estimated proportion of phylotypes in the environment represented in the library, ranged widely but on average was remarkably high and not correlated with library size. Phylotype richness was calculated by methods based on the frequency of occurrence of different phylotypes in 194 libraries that provided appropriate data. For 90% of aquatic-system libraries, and for 79% of non-aquatic libraries, the estimated phylotype richness was <200 phylotypes. Nearly all of the larger estimates were in aquatic sediments, digestive systems and soils. However, the approaches used to estimate phylotype richness may yield underestimates when libraries are too small. A procedure is described to provide an objective means of determining when a library is large enough to provide a stable and unbiased estimate of phylotype richness. A total of 56 libraries, including 44 from aquatic systems, were considered ‘large enough’ to yield stable estimates suitable for comparing richness among environments. Few significant differences in phylotype richness were observed among aquatic environments. For one of two richness estimators, the average phylotype richness was significantly lower in hyperthermal environments than in sediment and bacterioplankton, but no other significant differences among aquatic environments were observed. In general, and with demonstrated exceptions, published studies have captured a large fraction of bacterial diversity in aquatic systems. In most cases, the estimated bacterial diversity is lower than we would have expected, although many estimates should be considered minimum values. We suggest that on local scales, aquatic bacterial diversity is much less than any predictions of their global diversity, and remains a tractable subject for study. The global-scale diversity of aquatic Bacteria, on the other hand, may be beyond present capabilities for effective study.