Chapter 5 Genetic Analysis in Bacteria

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 5.2.1 Transfer by conjugation （结合传递） 5.2.2 Three transfer types (F+, F-, Hfr, F’ cells) 5.2.3 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 5.3.1 Bacteriophage and its properties 5.3.2 Generalized transduction （普通转导） 5.3.3 Specialized transduction （特定转导） 5.3.4 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

5.1 Plasmid patterns 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.

遗传物质的分布： E.coli: 大环DNA 主染色体 4×106bp 质粒小染色体，大环DNA的0.5%~2% ，约4000~5000bp E.coli encodes all the enzymes it needs for amino-acid and nucleotide biosynthesis, it can grow in minimal media, which containglucoseas the only source of carbon and energy, andinorganic saltsas the source of the other elements that compose bacterial cells.

Plasmid pattern 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)

Chapter 5 Genetic Analysis in Bacteria and Bacteriophages 第五章细菌与噬菌体遗传 5.1 Plasmid patterns 质粒类型 5.2 Transfer of genes between bacteria 细菌间基因的传递 5.2.1 Transfer by conjugation （结合传递） 5.2.2 Three transfer types (F+, F-, Hfr, F’ cells) 5.2.3 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 5.3.1 Bacteriophage and its properties 5.3.2 Generalized transduction （普通转导） 5.3.3 Specialized transduction （特定转导） 5.3.4 Recombination in bacteria

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） Strain A: met- bio-thr+ leu+ thi+ Strain B: met+ bio+thr- leu- thi- (营养缺陷型菌原养型完全培养基)

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-

5.2.2 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- •HIGHcross-overs and recombination between main DNA •F- F- flash F- Hfr

• Hfr F’ F- F’ integration –sexduction(性导) 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

• F- F’ Hfr •Low frequency

1) 2) 3) F’ 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?

Possible crosses between strains below, identify which strains are F+, F- and Hfr cell E1 E2 E3 E4 E5 C1 F O M F O C2 O M O O M C3 O F O O F C4 F O M F O C5 O F O O F O= none; F=few; M= many exconjugants

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

5.2.3 Interrupted mating experiment – 中断杂交技术 - by E. Wollman and E. Jacob in 1957 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.

Hfr strain (strsazir tonAr gal+ lac +) F- (strrazis tonAsgal- lac-) Hfr×F- 隔时取样振荡中断杂交接种 ( str. included to kill Hfr)strr F- genotype 9’ azir 11’ tonAr 18 lac + 25’ gal + % min

O azi tonA lac gal F 9 11 18 25 gene order and mapunit in Hfrcell

Application: • 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 pro 2 AB312 gly lac his pur gal 3

Chapter 5 Genetic Analysis in Bacteria and Bacteriophages 第五章细菌与噬菌体遗传 5.1 Plasmid patterns 质粒类型 5.2 Transfer of genes between bacteria 细菌间基因的传递 5.2.1 Transfer by conjugation （结合传递） 5.2.2 Three transfer types (F+, F-, Hfr, F’ cells) 5.2.3 Interrupted mating experiment – 中断杂交技术 5.3 Transfer of genes between bacterium and bacterialviruses 细菌与噬菌体间基因的传递 5.3.1 Bacteriophage and its properties 5.3.2 Generalized transduction （普通转导） 5.3.3 Specialized transduction （特定转导） 5.3.4 Recombination in bacteria

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

Temperate phageshave a choice between the lytic(溶菌) and an alternativelysogenic(溶原) pathway. The latter involves integration into the host’s chromosome where the phage, now termed aprophage (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 intransduction. 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

Chapter 5 Genetic Analysis in Bacteria