Simultaneous enumeration of different bacteria using reverse sample genome probing technique

Quantitative reverse sample genome probing (RSGP) with lambdaDNA as an internal standard was used to enumerate the total numbers of Rhizobium sp. CCRC 13560, Rhizobium meliloti CCRC 13516 and Bradyrhizobium sp. CCRC 13585. K(lambda)/Kx ratios varied between the three species but also in response to the amounts of lambdaDNA or genomic DNA used in the labeling mixture or fixed upon the membrane. Comparative enumerations of pure cultures revealed higher counts using genomic probing as compared with growth-based colony forming units (CFU; 3.4+/-1.7-fold higher for R. meliloti, 6.4+/-7.8-fold higher for Rhizobium sp. and 0.34+/-0.17-fold higher for Bradyrhizobium sp.). In mixed cultures, the estimated cell numbers using genomic probing were 126+/-172-, 85+/-83- and 4.0+/-3.4-fold higher (same respective order) than the growth-based assay. By replacing the klambda/kx ratio with k'lambda/k'x (slope from signal intensity of differently diluted lambdaDNA/slope from signal intensity of differently diluted target DNAxf(x)/flambda), significant improvement in the accuracy of the estimation was achieved. The calculated cell numbers via the genomic probe technique were 0.99+/-0.13-, 1.25+/-0.23- and 0.18+/-0.11-fold higher than the respective CFUs in pure cultures of R. meliloti, Rhizobium sp. and Bradyrhizobium sp. In samples containing mixed populations, the estimated numbers from genomic probing were 1.25+/-0.51-, 45.9+/-14.8- and 0.27+/-0.07-fold higher than the CFU-derived cell count (same respective order).     

Analysis of environmental microbial communities by reverse sample genome probing

Development of fast and accurate methods for monitoring environmental microbial diversity is one of the great challenges in microbiology today. Oligonucleotide probes based on 16S rRNA sequences are widely used to identify bacteria in the environment. However, the successful development of a chip of immobilized 16S rRNA probes for identification of large numbers of species in a single hybridization step has not yet been reported. In reverse sample genome probing (RSGP), labelled total community DNA is hybridized to arrays in which genomes of cultured microorganisms are spotted on a solid support in denatured form. This method has provided useful information on changes in composition of the cultured component of microbial communities in oil fields, the soil rhizhosphere, hydrocarbon-contaminated soils and acid mine drainage sites. Applications and limitations of the method, as well as the prospects of extending RSGP to cover also the as yet uncultured component of microbial communities, are evaluated.     

Quantitative reverse sample genome probing of microbial communities and its application to oil field production waters

[This corrects the article on p. 4101 in vol. 59.].     

Precision genome engineering in lactic acid bacteria

Innovative new genome engineering technologies for manipulating chromosomes have appeared in the last decade. One of these technologies, recombination mediated genetic engineering (recombineering) allows for precision DNA engineering of chromosomes and plasmids in Escherichia coli. Single-stranded DNA recombineering (SSDR) allows for the generation of subtle mutations without the need for selection and without leaving behind any foreign DNA. In this review we discuss the application of SSDR technology in lactic acid bacteria, with an emphasis on key factors that were critical to move this technology from E. coli into Lactobacillus reuteri and Lactococcus lactis. We also provide a blueprint for how to proceed if one is attempting to establish SSDR technology in a lactic acid bacterium. The emergence of CRISPR-Cas technology in genome engineering and its potential application to enhancing SSDR in lactic acid bacteria is discussed. The ability to perform precision genome engineering in medically and industrially important lactic acid bacteria will allow for the genetic improvement of strains without compromising safety.     

Towards high-throughput genome engineering in lactic acid bacteria

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Detection of Thiobacillus ferrooxidans in acid mine environments by indirect fluorescent antibody staining.

An indirect fluorescent antibody (FA) staining technique was developed for the rapid detection of Thiobacillus ferrooxidans. The specificity of the FA stain for T. ferrooxidans was demonstrated with both laboratory and environmental samples. Coal refuse examined by scanning electron microscopy exhibited a rough, porous surface, which was characteristically covered by water-soluble crystals. Significant numbers of T. ferrooxidans were detected in the refuse pores. A positive correlation between numbers of T. ferrooxidans and acid production in coal refuse in the laboratory was demonstrated with the FA technique.     

Detection of Thiobacillus ferrooxidans in acid mine environments by indirect fluorescent antibody staining.

An indirect fluorescent antibody (FA) staining technique was developed for the rapid detection of Thiobacillus ferrooxidans. The specificity of the FA stain for T. ferrooxidans was demonstrated with both laboratory and environmental samples. Coal refuse examined by scanning electron microscopy exhibited a rough, porous surface, which was characteristically covered by water-soluble crystals. Significant numbers of T. ferrooxidans were detected in the refuse pores. A positive correlation between numbers of T. ferrooxidans and acid production in coal refuse in the laboratory was demonstrated with the FA technique.     

The community structure of sessile heterotrophic bacteria stressed by acid mine drainage

Microbial communities that developed on glass slides suspended in acid-polluted (pH=2.9) and nonpolluted (pH=6.5) but otherwise similar waters showed evidence of stress when suspended at the opposite station. Glucose incorporation was inhibited in both translocated communities, but the inhibition was not as severe and recovery of activity was faster for the acid-developed community as compared to the circumneutral community. The communities contained a substantially different set of members with little overlap. The range of pH values at which the members of the acid-developed community could function suggested that the members of that community were generalists, as opposed to narrowly constrained members of the community from the circumneutral station. Based on the proportion of test characters that received positive responses, the organisms from the acidic site were more general in their abilities (47.6% positive) as compared with the neutral counterparts (18.7% positive). The results support the concept that communities developed in extreme environments tend to be generalists, whereas those from mesic environments, due to the higher levels of competition present, tend to be specialists. Furthermore, the study of microbial communities in dynamic systems such as streams and reservoir inflows is facilitated by the use of solid surfaces which allow an assemblage of nontransient microbes to develop.     

Identification of cellulolytic bacteria in soil by stable isotope probing

Plant residues, mainly made up of cellulose, are the largest fraction of organic carbon material in terrestrial ecosystems. Soil microorganisms are mainly responsible for the transfer of this carbon to the atmosphere, but their contribution is not accurately known. The aim of the present study was to identify bacterial populations that are actively involved in cellulose degradation, using the DNA-stable isotope probing (DNA-SIP) technique. ¹³C-cellulose was produced by Acetobacter xylinus and incubated in soil for 7, 14, 30 and 90 days. Total DNA was extracted from the soil, the ¹³C-labelled (heavy) and unlabelled (light) DNA fractions were separated by ultracentrifugation, and the structure of active bacterial communities was analysed by bacterial-automated ribosomal intergenic spacer analysis (B-ARISA) and characterized with denaturing gradient gel electrophoresis (DGGE). Cellulose degradation was associated with significant changes in bacterial community structure issued from heavy DNA, leading to the appearance of new bands and increase in relative intensities of other bands until day 30. The majority of bands decreased in relative intensity at day 90. Sequencing and phylogenetic analysis of 10 of these bands in DGGE profiles indicated that most sequences were closely related to sequences from organisms known for their ability to degrade cellulose or to uncultured soil bacteria.     

Analysis of methanotrophic bacteria in Movile Cave by stable isotope probing

Movile Cave is an unusual groundwater ecosystem that is supported by in situ chemoautotrophic production. The cave atmosphere contains 1-2% methane (CH4), although much higher concentrations are found in gas bubbles that keep microbial mats afloat on the water surface. As previous analyses of stable carbon isotope ratios have suggested that methane oxidation occurs in this environment, we hypothesized that aerobic methane-oxidizing bacteria (methanotrophs) are active in Movile Cave. To identify the active methanotrophs in the water and mat material from Movile Cave, a microcosm was incubated with a 10%13CH4 headspace in a DNA-based stable isotope probing (DNA-SIP) experiment. Using improved centrifugation conditions, a 13C-labelled DNA fraction was collected and used as a template for polymerase chain reaction amplification. Analysis of genes encoding the small-subunit rRNA and key enzymes in the methane oxidation pathway of methanotrophs identified that strains of Methylomonas, Methylococcus and Methylocystis/Methylosinus had assimilated the 13CH4, and that these methanotrophs contain genes encoding both known types of methane monooxygenase (MMO). Sequences of non-methanotrophic bacteria and an alga provided evidence for turnover of CH4 due to possible cross-feeding on 13C-labelled metabolites or biomass. Our results suggest that aerobic methanotrophs actively convert CH4 into complex organic compounds in Movile Cave and thus help to sustain a diverse community of microorganisms in this closed ecosystem.     

3种淡水环境中低核酸含量细菌的滤过性

【目的】增加低核酸含量(LNA)细菌与过滤性细菌之间的认识。【方法】采用流式细胞技术(FCM)、变性梯度凝胶电泳(DGGE)及统计学分析研究3种典型淡水环境中细菌群落与滤过性。【结果】LNA细菌与过滤性细菌在数量上具有很好的相关性(y=0.646x+22.42,R2=0.984,P0.01),细菌的滤过性不仅与细菌大小有关,还与细胞整体形状及其伸缩性有关;细菌群落组织与LNA细菌比重呈负相关性,而与HNA细菌呈正相关性。【结论】0.45μm膜过滤对水样微生物群落中的LNA细菌具有极强的筛选效果;细菌群落组织与其基于流式细胞技术测定的基因含量具有密切联系。     

Cultivation-independent detection of autotrophic hydrogen-oxidizing bacteria by DNA stable-isotope probing

Knallgas bacteria are a physiologically defined group that is primarily studied using cultivation-dependent techniques. Given that current cultivation techniques fail to grow most bacteria, cultivation-independent techniques that selectively detect and identify knallgas bacteria will improve our ability to study their diversity and distribution. We used stable-isotope probing (SIP) to identify knallgas bacteria in rhizosphere soil of legumes and in a microbial mat from Obsidian Pool in Yellowstone National Park. When samples were incubated in the dark, incorporation of (13)CO(2) was H(2) dependent. SIP enabled the detection of knallgas bacteria that were not detected by cultivation, and the majority of bacteria identified in the rhizosphere soils were betaproteobacteria predominantly related to genera previously known to oxidize hydrogen. Bacteria in soil grew on hydrogen at concentrations as low as 100 ppm. A hydB homolog encoding a putative high-affinity NiFe hydrogenase was amplified from (13)C-labeled DNA from both vetch and clover rhizosphere soil. The results indicate that knallgas bacteria can be detected by SIP and populations that respond to different H(2) concentrations can be distinguished. The methods described here should be applicable to a variety of ecosystems and will enable the discovery of additional knallgas bacteria that are resistant to cultivation.     

Advantages of sucrose-dependent extracellular polysaccharide production to lactic acid bacteria in sucrose-rich environments

G.A. DYKES, I. GEORNARAS AND A. VON HOLY. 1995. Some possible advantages of sucrosedependent extracellular polysaccharide production to Lactobacillus L191 from baker's yeast were investigated. A ca log 1 plaque-forming units ml^-1decrease in attachment of bacteriophage AB1 to cells of Lactobacillus L191 grown in the presence of sucrose as compared to cells grown in the absence of sucrose was noted. On the other hand, a ca log 1 colony-forming units cm^-2increase in attachment of Lactobacillus L191 to stainless steel surfaces when grown in the presence of sucrose compared to the absence of sucrose was observed. It was concluded that sucrose-dependent extracellular polysaccharide may provide a series of individually small but jointly synergistic selective advantages to strains producing it.     

Rapid sample processing and fast gas chromatography for identification of bacteria by fatty acid analysis

A commercially available system for microbial identification by fatty acid analysis (Microbial Identification System (MIS), MIDI, Newark, DE, USA) requires a four-step sample derivatization procedure in screw-cap test tubes. By using glass tubes in a 96-well format with multichannel pipetting, the time required for sample preparation can be greatly reduced. The standard gas chromatography column, 25 m long by 0.20 mm ID, is replaced with a 10 m long by 0.10 mm ID column, reducing the gas chromatography run time to one third of the standard time. Either or both of these procedures can be easily implemented in any laboratory using the MIDI system, resulting in faster identifications and higher sample throughput.     

CRISPR/Cas基因组编辑技术在乳酸菌中的应用及研究展望

乳酸菌是一类重要的食品工业微生物,目前对其功能基因鉴定和挖掘优良功能基因主要依赖于传统的基因同源重组技术,该技术尽管有较高的可靠性,但是存在操作繁琐、效率低下等不足,严重制约了乳酸菌优良菌株的遗传选育。CRISPR/Cas基因编辑技术极大提升了对多物种基因组的编辑效率,这为乳酸菌功能基因的快速鉴定及遗传改良提供了可能,但是现有的CRISPR/Cas基因编辑技术在乳酸菌的应用还存在诸多限制。本文综述了CRISPR/Cas基因编辑技术在乳酸菌基因组上的应用现状及亟待解决的问题,并展望了乳酸菌基因组编辑技术的未来发展趋势,为乳酸菌功能基因鉴定及遗传改良提供参考。     

Physiology of acetic acid bacteria in light of the genome sequence of Gluconobacter oxydans

Acetic acid bacteria are a distinct group of microorganisms within the family Acetobacteriaceae. They are characterized by their ability to incompletely oxidize a wide range of carbohydrates and alcohols. The great advantage of these reactions is that many substrates are regio- and stereoselectively oxidized. This feature is already exploited in several combined biotechnological-chemical procedures for the synthesis of sugar derivatives. Therefore, it is important to understand the basic concepts of this type of physiology to construct strains for improved or new oxidative fermentations. Based on the genome sequence of Gluconobacteroxydans, we will shed light on the central carbon metabolism, the composition of the respiratory chain and the analysis of uncharacterized oxidoreductases. In this context, the role of membrane-bound and -soluble dehydrogenases are of major importance in the process of incomplete oxidation. Other topics deal with the question of how these organisms generate energy and assimilate carbon. Furthermore, we will discuss how acetic acid bacteria thrive in their nutrient-rich environment and how they outcompete other microorganisms.     

Enhanced detection of surface-associated bacteria in indoor environments by quantitative PCR

Methods for detecting microorganisms on surfaces are needed to locate biocontamination sources and to relate surface and airborne concentrations. Research was conducted in an experimental room to evaluate surface sampling methods and quantitative PCR (QPCR) for enhanced detection of a target biocontaminant present on flooring materials. QPCR and culture analyses were used to quantitate Bacillus subtilis (Bacillus globigii) endospores on vinyl tile, commercial carpet, and new and soiled residential carpet with samples obtained by four surface sampling methods: a swab kit, a sponge swipe, a cotton swab, and a bulk method. The initial data showed that greater overall sensitivity was obtained with the QPCR than with culture analysis; however, the QPCR results for bulk samples from residential carpet were negative. The swab kit and the sponge swipe methods were then tested with two levels of background biological contamination consisting of Penicillium chrysogenum spores. The B. subtilis values obtained by the QPCR method were greater than those obtained by culture analysis. The differences between the QPCR and culture data were significant for the samples obtained with the swab kit for all flooring materials except soiled residential carpet and with the sponge swipe for commercial carpet. The QPCR data showed that there were no significant differences between the swab kit and sponge swipe sampling methods for any of the flooring materials. Inhibition of QPCR due solely to biological contamination of flooring materials was not evident. However, some degree of inhibition was observed with the soiled residential carpet, which may have been caused by the presence of abiotic contaminants, alone or in combination with biological contaminants. The results of this research demonstrate the ability of QPCR to enhance detection and enumeration of biocontaminants on surface materials and provide information concerning the comparability of currently available surface sampling methods.     

Detection of monochlorobenzene metabolizing bacteria under anoxic conditions by DNA-stable isotope probing

Cultivation-independent analyses were applied to study the structural diversity of the bacterial community which developed in groundwater inoculated microcosms actively metabolizing monochlorobenzene (MCB) under anaerobic conditions. Addition of ¹³C-labelled MCB demonstrated that the community produced ¹³CO₂ as a metabolite at slightly increasing rates over a period of 1,051 days while no ¹³C-methane evolved. Genetic profiles of partial 16S rRNA genes generated with the single-strand conformation polymorphism (SSCP) technique by PCR from directly extracted total DNA revealed that, despite the long incubation period, six replicate microcosms were characterized by almost the same microbial members. Nine distinguishable contributors to the SSCP-profiles were characterized by DNA sequencing, revealing the presence of different members from the phyla Proteobacteria, Fibrobacteres and from the candidate division OD1. DNA-stable isotope probing (SIP) was applied to distinguish the actual MCB metabolizing bacteria from the other community members. This study reveals for the first time the structural diversity of an anaerobic MCB metabolizing bacterial community. However, it also demonstrates the limitations of SIP to detect bacteria slowly metabolizing carbon sources under anaerobic conditions.     

利用内源CRISPR-Cas系统开展乳酸菌基因编辑的研究进展北大核心CSCD

CRISPR-Cas9技术是一种高效、精准的基因编辑工具,该技术的建立推动基因组编辑进入快速发展阶段。目前应用最广泛的Cas9蛋白是来源于酿脓链球菌(Streptococcuspyogenes)的SpyCas9,该蛋白作为“基因剪刀”在哺乳动物、植物等真核生物中应用较为广泛且成熟,但是该蛋白在一些乳酸菌中的应用仍然受到多种因素的限制。乳酸菌基因组上已发现多种类型的CRISPR系统,也蕴含着多种未表征的Cas蛋白,利用乳酸菌内源CRISPR-Cas系统,结合外源导入的向导RNA和同源修复模板,也可实现对乳酸菌基因组的编辑。这种基于内源CRISPR-Cas系统实现基因编辑的方式,具有打靶载体相对较小易转化、无外源Cas9蛋白对宿主细胞产生毒性等优势,相比于CRISPR-SpyCas9更适合于乳酸菌基因组进行编辑,可能是一些乳酸菌未来开展基因组编辑的主要手段,本文重点对此部分内容进行了综述。