Chapter 5 Genetic Analysis in Bacteria and Bacteriophages 第五章 细菌与噬菌体遗传 structure of bacterial genomes mechanisms by which bacteria transfer genes between cells of the same species mechanisms by which bacteria transfer genes between bacterial cells and bacterial viruses Geneticists can use their knowledge of bacterial genetics in general and of various forms of bacterial gene transfer in particular to identify, map, and characterize the genes that contribute to biological process.

5.1 Plasmid patterns 质粒类型 5.2 Transfer of genes between bacteria 细菌间基因的传递 Transfer by conjugation （ 结合传递） Three transfer types (F+, F-, Hfr, F’ cells) Interrupted mating experiment – 中断杂交技术 5.3 Transfer of genes between bacterium and bacterial viruses 细菌与噬菌体间基因的传递 Bacteriophage and its properties Generalized transduction （普通转导） Specialized transduction （特定转导） Recombination in bacteria

Advantages ： 1. Rapid reproduction, short life-span and large amount 2. Small genetic material, simple cell structure 3. Simple DNA structure, naked, helix and circular DNA 4. Monoploid DNA

Plasmids are chromosome-like structures found in bacterial cells, but which are not essential to bacterial growth. Some can move from one bacterium to another. The best studied is the F plasmid. This is an episome, an element that can replicate either independently or integrate into the bacterial chromosome. 5.1 Plasmid patterns

E.coli encodes all the enzymes it needs for amino-acid and nucleotide biosynthesis, it can grow in minimal media, which contain glucose as the only source of carbon and energy, and inorganic salts as the source of the other elements that compose bacterial cells. 遗传物质的分布： E.coli: 大环 DNA 主染色体 4×10 6 bp 质粒 小染色体，大环 DNA 的 0.5%~2% ， 约 4000~5000bp

 infectious ： from donor strain to recipient 从一个细菌（供体菌）转移到另一个不含质粒的受体菌中 non-infectious : 不能传递  replicable: isolated from chromosome 独位于染色体之外 combined: integrated into main chromosome and disengaged again 插入到主染色体上, 条件改变可脱离  F plasmid: F + - 供体菌 ( 雄性菌 ), 膜上有性须, 可转移 F - - 受体菌 ( 雌性菌 ), 无性须 R plasmid: antibiotic resistance ( 抗药性质粒 ) Plasmid pattern

5.1 Plasmid patterns 质粒类型 5.2 Transfer of genes between bacteria 细菌间基因的传递 Transfer by conjugation （ 结合传递） Three transfer types (F+, F-, Hfr, F’ cells) Interrupted mating experiment – 中断杂交技术 5.3 Transfer of genes between bacterium and bacterial viruses 细菌与噬菌体间基因的传递 Bacteriophage and its properties Generalized transduction （普通转导） Specialized transduction （特定转导） Recombination in bacteria Chapter 5 Genetic Analysis in Bacteria and Bacteriophages 第五章 细菌与噬菌体遗传

5.2 Transfer of genes between bacteria Focus on E.coli: the most studied and best understood species of bacteria is E.coli, a common inhabitant of vertebrate intestines. E.coli cells can grow in the complete absence of oxygen—the condition found in the intestines.

5.2.1 Transfer by conjugation （ 结合传递） ( 营养缺陷型菌 原养型 完全培养基 ) Strain A: met - bio - thr + leu + thi + Strain B: met + bio + thr - leu - thi -

Minimal medium

A met - bio - thr + leu + thi + B met + bio + thr - leu - thi - met + bio + thr + leu + thi + colonies

Founded by electronic microscope: Conjugation tube produced between two strains A B

Streptomycin ( 链霉素 ) treatment on A and B separately A x B Restrained reproduction colonies A x B Restrained reproduction no colony A B donor strain recipient F + F -

5.1 Plasmid patterns 质粒类型 5.2 Transfer of genes between bacteria 细菌间基因的传递 Transfer by conjugation （ 结合传递） Three transfer types (F+, F-, Hfr, F’ cells) Interrupted mating experiment – 中断杂交技术 5.3 Transfer of genes between bacterium and bacterial viruses 细菌与噬菌体间基因的传递 Bacteriophage and its properties Generalized transduction （普通转导） Specialized transduction （特定转导） Recombination in bacteria Chapter 5 Genetic Analysis in Bacteria and Bacteriophages 第五章 细菌与噬菌体遗传

5.2.2 Three transfer types: 1) 游离态 F 质粒 F + F - Low recombination between main chromosome 主染色体之间重组率低 F - F + flash

2) 结合态 F 质粒 F + integrated into mainDNA (Hfr strain) F - HIGH cross-overs and recombination between main DNA F - F - flash F- Hfr

3) F’ 质粒 When the F plasmid is excised from the bacterial chromosome it sometimes incorporates part of the chromosome into its own structure - F’ Hfr 菌株质粒从主染色体脱离时携带部分主染色体基因形成的 游离质粒 - F’ flash Hfr F’ F - F’ integration –sexduction ( 性导 )

Low frequency F- F’ Hfr

F’ 1) 2) 3) Interactive genetics – problem2

Applications: C Strain: arg - met + × E Strain: arg + met - arg + met + F + × F + Hfr× F - Hfr× F + F - × F - F + × F - Hfr× Hfr What type of crosses would give you many/few/no exconjugants?

C Strain: arg - met + × E Strain: arg + met - arg + met + F + × F + Hfr× F - Hfr× F + F - × F - F + × F - Hfr× Hfr What type of crosses would give you many/few/no exconjugants?

FOOFOC5 OFMOFC4 FOOFOC3 MOOMOC2 OFMOFC1 E5E4E3E2E1 Possible crosses between strains below, identify which strains are F +, F - and Hfr cell O= none; F=few; M= many exconjugants

FOOFOF+F+ OFMOFF-F- FOOFOF+F+ MOOMOHfr OFMOFF-F- F-F- F+F+ F-F- F+F+ Possible crosses between strains below, identify which strains are F +, F - and Hfr cell O= none; F=few; M= many exconjugants

5.1 Plasmid patterns 质粒类型 5.2 Transfer of genes between bacteria 细菌间基因的传递 Transfer by conjugation （ 结合传递） Three transfer types (F+, F-, Hfr, F’ cells) Interrupted mating experiment – 中断杂交技术 5.3 Transfer of genes between bacterium and bacterial viruses 细菌与噬菌体间基因的传递 Bacteriophage and its properties Generalized transduction （普通转导） Specialized transduction （特定转导） Recombination in bacteria Chapter 5 Genetic Analysis in Bacteria and Bacteriophages 第五章 细菌与噬菌体遗传

In the chromosome, plasmid can cause transfer of the entire chromosome to a recipient cell. The time at which each gene enters the recipient can be used to create a gene map Interrupted mating experiment – 中断杂交技术 - by E. Wollman and E. Jacob in 1957

Hfr ×F - 隔时取样 振荡 中断杂交 接种 ( str. included to kill Hfr) str r F - genotype 9’ azi r 11’ tonA r 18 lac + 25’ gal + Hfr strain (str s azi r tonA r gal + lac + ) F - (str r azi s tonA s gal - lac - ) min %

O azi tonA lac gal F gene order and mapunit in Hfr cell

Application: Hfr strains Transfer order of genes H AB312 0 thr pro lac pur gal his gly thi 0 thr thi gly his gal pur lac pro 0 pro thr thi gly his gal pur lac 0 pur lac pro thr thi gly his gal 0 thi thr pro lac pur gal his gly Draw the structure of main DNA Mark the transfer origin and the direction on it Mark the random integrated positions of F plasmid

H thr 1 thi gly his gal 3 pur lac pro 2 AB312 H AB312 0 thr pro lac pur gal his gly thi 0 thr thi gly his gal pur lac pro 0 pro thr thi gly his gal pur lac 0 pur lac pro thr thi gly his gal 0 thi thr pro lac pur gal his gly

5.1 Plasmid patterns 质粒类型 5.2 Transfer of genes between bacteria 细菌间基因的传递 Transfer by conjugation （ 结合传递） Three transfer types (F+, F-, Hfr, F’ cells) Interrupted mating experiment – 中断杂交技术 5.3 Transfer of genes between bacterium and bacterial viruses 细菌与噬菌体间基因的传递 Bacteriophage and its properties Generalized transduction （普通转导） Specialized transduction （特定转导） Recombination in bacteria Chapter 5 Genetic Analysis in Bacteria and Bacteriophages 第五章 细菌与噬菌体遗传

5.3.1 Bacteriophage and its properties Phages can be divided into two classes: virulent or temperate depending on how they behave after infection of a bacterium. when a virulent phage infects a bacterium it takes over the synthetic machinery of the host and uses it as a factory for the production of new phages. 5.3 Transfer of genes between bacterium and bacterial viruses

Temperate phages have a choice between the lytic ( 溶菌 ) and an alternative lysogenic ( 溶原 ) pathway. The latter involves integration into the host’s chromosome where the phage, now termed a prophage ( 原噬菌体 ), is dormant and replicates along with the rest of the host chromosome. Occasionally with low frequency the prophage loses its dormancy and converts to the lytic, replicating phages and lysing the bacterium.

溶原 / 菌过程

5.3.2 Generalized transduction （普通转导） Bacteriophages are involved in transduction. Virulent phages replicate in the bacterium. An occasional phage particle will contain a small fragment of bacterial DNA rather than bacteriophage DNA. The bacteriophage will transfer it to a recipient cell - generalized transduction 侵染 复制 宿主 DNA 断裂 噬菌体 DNA 组 装外壳，宿主 DNA 碎片随机装入 子噬菌体释放 带有原宿主 DNA 基因 侵染细菌 原宿主 DNA 片段与受体菌同源互补 部分二倍体形 成或发生交换重组 - 普通转导.

5.3.3 Specialized transduction （特定转导） Lysogenic prophage loses its dormant state and excised from the host DNA. Rarely the excision process is inaccurate, the phages carry and transfer host genes between bacteria – specialized transduction 结合态噬菌体切离宿主 DNA ，携带部分宿主基因，再 次侵染时与受体菌 DNA 同源互补及交换重组 。

5.3.4 Recombination in bacteria

Transformation - 转化 细菌细胞从周围介质吸收不同基因型细胞 的 DNA ，其基因型和表型发生改变的过程 Transfection – 转染 用除去蛋白质外壳的病毒核酸感染细胞或 原生质体的过程。转化的一种特殊形式 Transduction – 转导 以噬菌体为媒介，将细菌基因导入另一细 菌的过程。普通转导、特定转导