Methods to study microorganisms

in the environment

PART 1: Cultivation-based techniques (Chapter 10)

- how to identify and enumerate microorganisms?
PART 2: (Eco)physiological methods (Chapter 11)
- how to measure microbial activities (functions)?
PART 3: Tracking specific microorganisms (Chapter 13)
- how to track presence/activity of specific microorganisms?
PART 4: Nucleic acid based techniques (Chapter 13)
- how to identify and enumerate microorganisms?
1
Lecture 4 (last time)
Soil and Water Pollution – Experimental Assessment
Kristian K. Brandt

Methods to study microbial communities in

polluted environments

PART 1: Cultivation-based techniques (Chapter 10)

Enrichment and isolation of microorganisms
Viable counts and MPN enumeration of bacteria
The ‘great plate count anomaly’ and the uncultured majority
New approaches for enhanced or targeted cultivation of bacteria

2
 基于核酸的技术（第 13 章） 从环境中
Lecture 5 提取核酸 荧光原位杂交 (FISH) 群落分
Soil and Water Pollution – Experimental Assessment析 PCR 依赖群落分析（多种方法） 元
基因组学、元转录组学、元蛋白质组
Kristian K. Brandt 学和环境代谢组学不在此处涵盖（其
他几门课程）在哥本哈根大学）。

例如。高级微生物生物技术（区块 1

Methods to study microbial communities in

）和应用微生物学（区块 2）

polluted environments

PART 4: Nucleic acid based techniques (Chapter 13)

Nucleic acid extraction from the environment
Fluorescent in situ hybridization (FISH) community analysis
PCR dependent community analysis (wide variety of approaches)

Metagenomics, metatranscriptomics, metaproteomics, and

environmental metabolomics are NOT covered here
(several other courses at Univ of Copenhagen).
E.g. Advanced Microbial Biotechnology (Block 1) and Applied
Microbiology (Block 2)
3
 Nucleic acid extraction from
environmental samples
直接提取 DNA 或 RNA – 原位裂解细
胞，例如通过珠磨、冻融循环、洗涤
剂应用等） – 去除细胞碎片和土壤颗

• Direct DNA or RNA extraction

粒 – 核酸沉淀 – 进一步纯化

– In situ lysis of cells e.g. by bead

beating, freeze-thaw cycles,
detergent application etc.)
– Removal of cell debris and soil
particles
– Precipitation of nucleic acids
– Further purification
从样品中提取细胞（例如 Nycodenz 离
心）和随后从细胞中提取 DNA

• Cell extraction from sample

(e.g. Nycodenz centrifugation)
and subsequent DNA
extraction from cells Purification 4
Nycodenz extraction of soil bacteria
(large non-sheared DNA fragments can be obtained)

Nycodenz density gradient centrifugation

1.3 g/mL

5
Nybroe et al., from Modern soil microbiology 2007.
In situ lysis relative to cell extraction method

• Advantages
– Higher DNA yield
– More representative community sampling
– Fungal DNA also obtained
– Rapid 相对于细胞提取方法的原位裂解 优点
– 更高的 DNA 产量 – 更具代表性的
群落采样 – 还获得了真菌 DNA – 快
速 缺点 – 更多的 DNA 剪切（更小的
DNA 片段） – 更多的腐殖质污染物

• Disadvantages （可能导致 PCR 抑制）

– More shearing of DNA (smaller DNA fragments)

– More humic contaminants (can lead to PCR inhibition)

6
 Base pairing in DNA
DNA sequences are determined by four bases (A – G – T – C)
A-T and G-C base pairing forms the basis for DNA hybridization
techniques and Polymerase Chain Reaction (PCR)
DNA 中的碱基配对 DNA 序列由四个
碱基 (A – G – T – C) 决定 A-T 和 G-
C 碱基配对构成了 DNA 杂交技术和聚
合酶链式反应 (PCR) 的基础

7
The 16S rRNA
molecule
RNA 序列由相同的“基本语言”确定
，只是胸腺嘧啶 (T) 被尿嘧啶 (U) 取
代（A-U 和 G-C 碱基配对） rRNA 由
保守区和可变区 (V1-V9) 组成
RNA sequences are determined
by the same “base language”
except that thymine (T) is replaced
by uracil (U)
(A-U and G-C base pairing)

rRNA consist of conserved and

variable regions (V1-V9)

rRNA sequence is the “gold

standard” for taxonomic
identification of prokaryotes (can
resolve different genera, but
cannot differentiate closely related
rRNA序列是原核生物分类鉴定的“金
species) 标准”（可分辨不同属，但不能区分
近缘种）单链DNA可与单链RNA通过
Single stranded DNA can 碱基配对杂交

hybridize with single stranded

RNA by base pairing
Figure 12.12
 Fluorescent In Situ Hybridization (FISH)
寡核苷酸 DNA 探针用合适的荧光染料 Since FISH analyses are conducted in situ, an inher
标记 探针将与具有互补序列的 rRNA ent dif-
杂交 系统发育 FISH 染色因此可以基 ficulty of this technique is differentiating signal from
background noise such as probe nonspecifically so
于 16S rRNA 序列相似性靶向特定的分 rbed to
类群，并允许通过荧光显微镜对微生 soil particles or the presence of naturally fluorescing
物进行原位可视化 com-
pounds including metals and some soil minerals.
• Oligonucleotide DNA probe is tagged
with a suitable fluorescent dye
• The probe will hybridize to rRNA with
complementary sequence
• Phylogenetic FISH staining therefore
can target specific taxonomic groups
based on 16S rRNA sequence similarity
and allows for in situ visualization of
microorganisms by fluorescence
microscopy
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 PCR dependent community analysis PCR 依赖性群落分析 小亚基 rRNA 基
因（单基因）方法 – 直接 DNA 提取
– 小亚基 rRNA 基因的 PCR 扩增（例
• Small subunit rRNA gene (single gene) 如

approach 细菌 16S rRNA 基因）使用或多或少的

特异性引物（PCR 特异性由引物序列
决定） – PCR 后 DNA 分析（许多替
– Direct DNA extraction 代方法） DNA 指纹技术 测序和系统
发育分析 – 高通量测序允许对社区结

– PCR amplification of small-subunit rRNA gene (e.g.

构进行全面的社区分析和多样性（物
种丰富度）

bacterial 16S rRNA gene) using more or less specific

primers (PCR specificity determined by primer
sequences)
– Post PCR DNA analysis (many alternatives)
• DNA fingerprinting techniques
• Sequencing and phylogenetic analysis
– High-throughput sequencing allows for comprehensive community
analysis of community structure and diversity (species richness)
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 The Polymerase Chain Reaction (PCR)
• PCR is basically DNA replication in a test
tube
– Conceived for laboratory use by Kary Mullis in
1984-1985 (Nobel Prize in 1993)
– Also called DNA amplification
聚合酶链式反应 (PCR) PCR 基本上是
试管中的 DNA 复制 – 由 Kary Mullis
于 1984-1985 年设想用于实验室（1993
年获得诺贝尔奖） – 也称为 DNA 扩
增

© 2012 Pearson Education, Inc.

 Steps in PCR
• Prepare PCR master mix containing all PCR
reagents:
– Buffer, water, primers, dNTP’s, and heat-stable
DNA polymerase
• Add template DNA to be PCR amplified
(=DNA extracted from environmental sample)
• Heat and cool (many cycles)
PCR 步骤 准备包含所有 PCR 试剂的 P
CR 预混液： – 缓冲液、水、引物、d
NTP 和热稳定 DNA 聚合酶 添加模板
DNA 进行 PCR 扩增（=从环境样品中
提取的 DNA） 加热和冷却（许多周
期）

© 2012 Pearson Education, Inc.

PCR cycling: Denaturation (e.g. 95 °C) + Primer annealing (e.g. 55-62 °C) + Primer extension/elongation (72 °C)
Copies of
PCR target
cycle sequence
Target sequence

0 1
DNA
Heat Primers
polymerase

Primer extension

1 2 108

Copies of target sequence

107
106
105
104
103
102
Repeat cycle
2 4 10

2 4 6 8 10 12 14 16 18 20
Repeat cycle 3 8 Number of PCR cycles

© 2012 Pearson Education, Inc.

Standard PCR dependent approaches for
cultivation-independent community
analysis
用于独立于培养的群落分析的标准 PC
R 依赖方法

Sequencing is replacing DNA

fingerprinting techniques

14
© 2012 Pearson Education, Inc.
Sequencing has replaced fingerprinting techniques
for community analysis

15
Barcoded pyrosequencing workflow
(‘pyro tagging’) 条形码焦磷酸测序工作流程（“pyro
标记”）

16
Hamady & Knight (2009) Genome Research
 微生物群落多样性通常基于 16S rRNA

Microbial community diversity is often based

基因序列分析

on 16S rRNA gene sequence analysis

• Phylotype: an operational taxonomic unit (OTU) defined
with reference to rRNA sequence similarity
– E.g. at least >97 % or >99 % similarity
• Thousands of unique phylotypes per g of soil, but true
species richness is higher, but controversial (bacterial
species definition is vague)
• Much higher diversity in soils and sediments as compared
to pelagic environments
• cDNA derived from 16S rRNA may be analyzed in parallel
种系型：参照 rRNA 序列相似性定义
的操作分类单元 (OTU) – 例如至少 >
97% 或 >99% 的相似性 每克土壤有数
千种独特的种系型，但真正的物种丰
富度更高，但存在争议（细菌物种定
义模糊） 与远洋环境相比，土壤和沉
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积物的多样性更高 来自 16S rRNA 的 c
Detected bacterial community composition
depends on the used approach
检测到的细菌群落组成取决于使用的
方法

Comparison of two DNA extraction kits

from Qiagen (a commercial kit provider)

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 Types of PCR
• Conventional (end-point) PCR 常规（终点）PCR – 标准 PCR 一对
– Standard PCR 引物靶向一个模板 – 多重 PCR
引物靶向多个模板
多对

• One template targeted by one primer pair

– Multiplex PCR
• Multiple templates targeted by multiple primer pairs

• Quantitative real-time PCR (qPCR)

– PCR product is detected in real-time using
fluorescent probes allowing for quantification
of gene abundances (see Figure 13.7)
– High-throughput qPCR chips allow for parallel
quantification of many genes
定量实时 PCR (qPCR) – 使用荧光探
针实时检测 PCR 产物，允许对基因丰
度进行定量（见图 13.7） – 高通量 qP
CR 芯片允许对许多基因进行平行定量
19
Templates for PCR used in cultivation-
independent community analysis 用于培养独立群落分析的 PCR 模板
群落 DNA – 表示群落中目标基因的
存在（如果每个基因组的基因拷贝数
• Community DNA 已知，则等于目标生物体的存在）
– Indicates the presence of the target gene in the community (equals
presence of target organism if gene copy number per genome is
known). Target gene may be 16S rRNA gene or a functional gene
encoding a protein (e.g. catabolic genes involved in pollutant
degradation). 。目标基因可能是 16S rRNA 基因或编
码蛋白质的功能基因（例如参与污染
物降解的分解代谢基因）
• Complementary DNA (cDNA) derived from community 16S rRNA
– ‘Biased’ toward identification of the active organisms (the number of
ribosomes increases with increasing growth activity, but ribosome
number per cell also depend on other factors). 来自社区 16S rRNA 的互补 DNA (cDN
A) – “偏向”识别活性生物（核糖体
的数量随着生长活动的增加而增加，
但每个细胞的核糖体数量也取决于其
• cDNA derived from community mRNA 他因素）。

– Indicates the presence of actively transcribed target genes (e.g.

catabolic genes involved in pollutant degradation).
来自社区 mRNA 的 cDNA – 表示存在
活跃转录的目标基因（例如 20

参与污染物降解的分解代谢基因）。
Lecture 4
Soil and Water Pollution – Experimental Assessment
Kristian K. Brandt

Methods to study microbial communities in

polluted environments
（生态）生理学方法（第 11 章）

PART 3: (Eco)physiological methods (Chapter 11)

Microbial functions
Linking microbial functions to specific organisms (Lecture 5)

21
Linking microbial functions to
specific organisms
将微生物功能与特定生物联系起来

Who is doing what?

22
 Trophic interactions in the microbial world
– Who ate the bananas?

在这种情况下，香蕉等于生长基质，
它可能是有机污染物（例如特定化合
物或同位素标记的生物质）

Bananas in this case equal a growth substrate,

which may be an organic pollutant
(e.g. a specific compound or isotope labeled biomass)
23
Linking specific genes and functions to
specific organisms

• Stable isotope probing (SIP): links specific

metabolic activity to taxonomic identification
using a stable isotope
– Microorganisms metabolizing stable isotope (e.g.,
13C) incorporate it into their DNA

• DNA with 13C can then be used to identify the

organisms that metabolized the 13C
– RNA-SIP is also possible 稳定同位素探测 (SIP)：使用稳定同位
素将特定代谢活动与分类学鉴定联系
起来
– 代谢稳定同位素（例如 13C）的微
生物将其整合到其 DNA 中 带有 13C
的 DNA 然后可用于鉴定代谢 13C 的生
物体
– RNA-SIP 也是可能的 24
 DNA-SIP flow chart
13C-DNA

This cell
metabolizes
13C substrate

12C-DNA

Feed 13C Extract Separate Remove and

substrate DNA light (12C) 13C-DNA analyze (PCR
from heavy 16S rRNA or

Colin Murrell
Environmental These cells do not (13C) DNA metabolic genes,
sample metabolize 13C or do genomics)
substrate 12C-DNA

Ultracentrifuge
tube with DNA

25
© 2012 Pearson Education, Inc.
 SIP outline
用 13C 标记的底物孵育土壤样品

Incubate soil sample with 13C-

DNA-SIP labelled substrate rRNA-SIP

Extract DNA Extract rRNA

Isopycnic centrifugation Isopycnic centrifugation

(density gradient) 等密度离心（密度梯度） (density gradient)

Gradient fractionation into Gradient fractionation into

‘light’ and ‘heavy’ DNA 梯度分离成“轻”和“重”DNA
‘light’ and ‘heavy’ rRNA

PCR Reverse Transcription PCR

逆转录聚合酶链反应
DNA characterization
(fingerprinting, sequencing,
phylogenetic analysis etc.)
DNA 表征（指纹图谱、测序、系统发
育分析等） 26
 Part 4: Take home messages
• New molecular biology methods (e.g. next-generation
sequencing and bioinformatical tools) are transforming
environmental microbiology 新的分子生物学方法（例如下一代测
序和生物信息学工具）正在改变环境
微生物学 所有微生物群落分析方法都
存在一定程度的偏差，但有些方法比
其他方法更偏
• All methods for microbial community analysis are biased
to a certain extent, but some more than others

• The molecular tool box is continuously being expanded

(only “the tip of the iceberg” is covered in this lecture)
分子工具箱正在不断扩展（本讲座仅
涵盖“冰山一角”） 微生物群落分析
推荐使用多相方法（即并行使用多种
方法）
• Polyphasic approach (i.e. parallel use of multiple
methods) is recommended for microbial community
analysis 27