Faruque Shah M, Kamruzzaman M, Nandi Ranjan K, Ghosh A N, Nair G Balakrish, Mekalanos John J, Sack David A
Molecular Genetics Laboratory, International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka 1212, Bangladesh.
Infect Immun. 2002 Jan;70(1):163-70. doi: 10.1128/IAI.70.1.163-170.2002.
In toxigenic Vibrio cholerae, cholera toxin is encoded by the CTX prophage, which consists of a core region carrying ctxAB genes and genes required for CTXPhi morphogenesis, and an RS2 region encoding regulation, replication, and integration functions. Integrated CTXPhi is often flanked by another genetic element known as RS1 which carries all open reading frames (ORFs) found in RS2 and an additional ORF designated rstC. We identified a single-stranded circularized form of the RS1 element, in addition to the CTXPhi genome, in nucleic acids extracted from phage preparations of 32 out of 83 (38.5%) RS1-positive toxigenic V. cholerae strains analyzed. Subsequently, the corresponding double-stranded replicative form (RF) of the RS1 element was isolated from a representative strain and marked with a kanamycin resistance (Km(r)) marker in an intergenic site to construct pRS1-Km. Restriction and PCR analysis of pRS1-Km and sequencing of a 300-bp region confirmed that this RF DNA was the excised RS1 element which formed a novel junction between ig1 and rstC. Introduction of pRS1-Km into a V. cholerae O1 classical biotype strain, O395, led to the production of extracellular Km(r) transducing particles, which carried a single-stranded form of pRS1-Km, thus resembling the genome of a filamentous phage (RS1-KmPhi). Analysis of V. cholerae strains for susceptibility to RS1-KmPhi showed that classical biotype strains were more susceptible to the phage compared to El Tor and O139 strains. Nontoxigenic (CTX(-)) O1 and O139 strains which carried genes encoding the CTXPhi receptor toxin-coregulated pilus (TCP) were also more susceptible (>1,000-fold) to the phage compared to toxigenic El Tor or O139 strains. Like CTXPhi, the RS1Phi genome also integrated into the host chromosomes by using the attRS sequence. However, only transductants of RS1-KmPhi which also harbored the CTXPhi genome produced a detectable level of extracellular RS1-KmPhi. This suggested that the core genes of CTXPhi are also required for the morphogenesis of RS1Phi. The results of this study showed for the first time that RS1 element, which encodes a site-specific recombination system in V. cholerae, can propagate horizontally as a filamentous phage, exploiting the morphogenesis genes of CTXPhi.
在产毒素霍乱弧菌中，霍乱毒素由CTX噬菌体编码，该噬菌体由一个携带ctxAB基因和CTXΦ形态发生所需基因的核心区域，以及一个编码调控、复制和整合功能的RS2区域组成。整合的CTXΦ通常两侧是另一个称为RS1的遗传元件，它携带RS2中发现的所有开放阅读框（ORF）和一个额外的ORF，即rstC。在分析的83株RS1阳性产毒素霍乱弧菌菌株中的32株（38.5%）的噬菌体提取物中，除了CTXΦ基因组外，我们还鉴定出了RS1元件的单链环化形式。随后，从一株代表性菌株中分离出RS1元件相应的双链复制形式（RF），并在一个基因间位点用卡那霉素抗性（Km(r)）标记进行标记，构建pRS1-Km。对pRS1-Km的限制性内切酶分析和PCR分析以及对一个300bp区域的测序证实，这种RF DNA是切除的RS1元件，它在ig1和rstC之间形成了一个新的连接。将pRS1-Km导入霍乱弧菌O1古典生物型菌株O395，导致产生细胞外Km(r)转导颗粒，其携带单链形式的pRS1-Km，因此类似于丝状噬菌体（RS1-KmΦ）的基因组。对霍乱弧菌菌株对RS1-KmΦ的敏感性分析表明，与埃尔托生物型和O139菌株相比，古典生物型菌株对该噬菌体更敏感。携带编码CTXΦ受体毒素共调节菌毛（TCP）基因的非产毒素（CTX(-)）O1和O139菌株与产毒素埃尔托生物型或O139菌株相比，对该噬菌体也更敏感（>1000倍）。与CTXΦ一样，RS1Φ基因组也通过使用attRS序列整合到宿主染色体中。然而，只有同时携带CTXΦ基因组的RS1-KmΦ转导子才能产生可检测水平的细胞外RS1-KmΦ。这表明CTXΦ的核心基因也是RS1Φ形态发生所必需的。本研究结果首次表明，在霍乱弧菌中编码位点特异性重组系统的RS1元件可以作为丝状噬菌体水平传播，利用CTXΦ的形态发生基因。
