Accumulation of single-stranded DNA in Escherichia coli carrying the colicin plasmid pColE3-CA38.

We sequenced the complete 7118 bp circular plasmid pColE3-CA38 (pColE3) from Escherichia coli, located the previously identified colicin components together with two new ORFs that have homology to mobilization and transfer proteins, and found that pColE3 is highly similar to a plasmid present in enterohemorrhagic E. coli O111. We also found that unusual aspects of the plasmid include the inability to be completely digested with restriction endonucleases and asymmetric Phred DNA sequencing quality scores, with significantly lower scores in the forward direction relative to the colicin and immunity proteins consistent with plus (+) strand DNA. Comparing the A260 with picogreen double-stranded DNA (dsDNA) fluorescence and oligreen single-stranded DNA (ssDNA) fluorescence as well as metachromatic staining by acridine orange, we found that the undigested pColE3 DNA stains preferentially as ssDNA and that it coexists with dsDNA. We also identified ssDNA in pColE5 and pColE9 but not in pColE1. Colicin plasmids producing ssDNA may represent a new subclass of rolling-circle replication plasmids and add to the known similarities between colicins and filamentous phage.

msbB deletion confers acute sensitivity to CO2 in Salmonella enterica serovar Typhimurium that can be suppressed by a loss-of-function mutation in zwf.

BACKGROUND: Pathogens tolerate stress conditions that include low pH, oxidative stress, high salt and high temperature in order to survive inside and outside their hosts. Lipopolysaccharide (LPS), which forms the outer-leaflet of the outer membrane in Gram-negative bacteria, acts as a permeability barrier. The lipid A moiety of LPS anchors it to the outer membrane bilayer. The MsbB enzyme myristoylates the lipid A precursor and loss of this enzyme, in Salmonella, is correlated with reduced virulence and severe growth defects that can both be compensated with extragenic suppressor mutations. RESULTS: We report here that msbB (or msbB somA) Salmonella are highly sensitive to physiological CO2 (5%), resulting in a 3-log reduction in plating efficiency. Under these conditions, msbB Salmonella form long filaments, bulge and lyse. These bacteria are also sensitive to acidic pH and high osmolarity. Although CO2 acidifies LB broth media, buffering LB to pH 7.5 did not restore growth of msbB mutants in CO2, indicating that the CO2-induced growth defects are not due to the effect of CO2 on the pH of the media. A transposon insertion in the glucose metabolism gene zwf compensates for the CO2 sensitivity of msbB Salmonella. The msbB zwf mutants grow on agar, or in broth, in the presence of 5% CO2. In addition, msbB zwf strains show improved growth in low pH or high osmolarity media compared to the single msbB mutant. CONCLUSION: These results demonstrate that msbB confers acute sensitivity to CO2, acidic pH, and high osmolarity. Disruption of zwf in msbB mutants restores growth in 5% CO2 and results in improved growth in acidic media or in media with high osmolarity. These results add to a growing list of phenotypes caused by msbB and mutations that suppress specific growth defects.

Tumor-targeted Salmonella expressing cytosine deaminase as an anticancer agent.

The study was designed to evaluate whether TAPET-CD, an attenuated strain of Salmonella typhimurium expressing Escherichia coli cytosine deaminase (CD), was capable of converting nontoxic 5-fluorocytosine (5-FC) to the active antitumor agent 5-fluorouracil (5-FU). The antitumor effect of TAPET-CD plus 5-FC against subcutaneously implanted colon tumors was also evaluated. TAPET-CD was given to tumor-bearing mice by a single bolus intravenous administration followed with 5-FC by intraperitoneal administration. TAPET-CD accumulated in tumors at levels 1000-fold higher than that in normal tissues and high levels of 5-FU were detected in tumors in mice treated with both TAPET-CD and 5-FC. No 5-FU could be detected in normal tissues. Inhibition of tumor growth was observed in mice treated with either TAPET-CD alone or TAPET-CD in combination with 5-FC (TAPET-CD/5-FC), but not with 5-FC alone. TAPET-CD/5-FC inhibited tumor growth by 88%-96%, compared to TAPET-CD alone, which inhibited tumor growth by 38%-79%. These data suggest that tumor-targeting Salmonella could be used to deliver prodrug-converting enzyme selectively to tumors and produced anti-tumor effects when the corresponding prodrug was also given.