# Constitutive Expression of a Cytotoxic Anticancer Protein in Tumor-Colonizing Bacteria
## 3 Results  
### Fate of E. coli Injected into Tumor-Bearing Mice  
The fate of bacteria injected into tumor-bearing mice through the tail vein was examined using a common laboratory strain of _E. coli_, MG1655. _E. coli_ MG1655 (1 \(\times\) 10\({}^{8}\) CFU/mouse) was injected into BALB/c meeting bearing CT26 colon cancer xenograft tumors. At the indicated times after the injection, bacterial numbers were counted in the RES, in the liver and spleen, and in tumors using plating methods (Figure 1A). At 2 h after the injection, there were approximately 1 \(\times\) 10\({}^{7}\) CFU in the RES, and this number decreased gradually in a time-dependent manner, reaching approximately 5 \(\times\) 10\({}^{4}\) CFU at 120 h. The bacterial number in tumors was 1 \(\times\) 10\({}^{4}\) CFU at 2 h after the injection, and this increased to approximately 5 \(\times\) 10\({}^{8}\) CFU at 72 h. This result indicates that although \(\sim\)0.01% of the injected bacteria accumulated in tumor tissues initially, the immunocompromised environment allowed substantial proliferation of those bacteria, whereas those in the RES were cleared presumably by phagocytic immune cells (see below). Among the most activated genes the most highly induced was the rrmB operon, consisting of the transcription unit rrsB-gltT-rrflB-rrflB encoding the three major rRNA building blocks of ribosomes [32]. The expression profile of the rrmB operon (number of reads) was determined by RNA sequencing (Figure 1B). The normalized read coverages in the liver and tumor were 58,220 and 1,148,213 reads at 1 dpi, respectively. At 3 dpi, these values changed drastically because of a shortfall of reads in the liver, showing 67 and 1,780,236 reads for the liver and tumor, respectively. We hypothesized that the reads on day 1 in the liver were remnants of those from overnight culture. By day 3, the bacteria in the liver were perishing as rrmB expression ceased, whereas those in the tumor proliferated. To confirm these findings, we measured the activity of the rrmB P1 promoter, which is the major promoter driving the rrmB operon. This promoter is active during the early exponential phase of growth, when ribosomesare needed most, and declines sharply thereafter during the stationary phase [33]. Three Fis-binding sites in the upstream activation region are responsible for the activation of the rrnB P1 promoter (Figure 1C) [28]. A gene reporter system was constructed using the unstable GFP variant gfpOVA [29], which was cloned downstream of rrnB P1 in pBR322, generating prrnBP1-gfpOVA. _E. coli_ transformed with this plasmid were used to monitor rrnB P1 activity. During growth in vitro (Figure S2A), fluorescence intensity determined at 488-522 nm indicated activation of rrnB P1 during the exponential phase of growth in LB medium, in agreement with the results of qPCR analysis of gfpOVA structural RNA. Then, we attempted to determine rrnB P1 activity in the _E. coli_ injected into tumor-bearing mice using a fluorescence microscope; however, this failed due to weak emission of fluorescence. Alternatively, we measured rrnB P1 activity by qPCR analysis of the gfpOVA structural RNA relative to rpoB RNA, which is maintained at constant levels (Figure 1D) [34]. The activity of rrnB P1 increased by up to 40-fold in the bacteria in tumor tissues at 120 h, whereas those in the liver and spleen decreased over time. This result supports that those bacteria in tumor tissues proliferated, whereas those in the RES perished. We did not quantify the rRNA from the genomic rrnB operon because there are seven rrn operons in _E. coli_ with similar sequences, and the ribosomal RNAs that provide the foundation for ribosomes are extremely stable. Figure 1: Fate of _E. coli_ injected into tumor-bearing mice. BALB/c mice grafted with CT26 colon carcinoma cells received intravenous injection of _Escherichia coli_ MG1655 (\(n=4\) per group, \(1\times 10^{8}\) CFU/mouse) when tumor volumes reached 130–150 mm\({}^{3}\).