Search

A modification to Performance-Tested MethodSM 010403, GeneQuence Listeria Test (DNAH method), is described. The modified method uses a new media formulation, LESS enrichment broth, in single-step enrichment protocols for both foods and environmental sponge and swab samples. Food samples are enriched for 2730 h at 30C, and environmental samples for 2448 h at 30C. Implementation of these abbreviated enrichment procedures allows test results to be obtained on a next-day basis. In testing of 14 food types in internal comparative studies with inoculated samples, there were statistically significant differences in method performance between the DNAH method and reference culture procedures for only 2 foods (pasteurized crab meat and lettuce) at the 27 h enrichment time point and for only a single food (pasteurized crab meat) in one trial at the 30 h enrichment time point. Independent laboratory testing with 3 foods showed statistical equivalence between the methods for all foods, and results support the findings of the internal trials. Overall, considering both internal and independent laboratory trials, sensitivity of the DNAH method relative to the reference culture procedures was 90.5. Results of testing 5 environmental surfaces inoculated with various strains of Listeria spp. showed that the DNAH method was more productive than the reference U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) culture procedure for 3 surfaces (stainless steel, plastic, and cast iron), whereas results were statistically equivalent to the reference method for the other 2 surfaces (ceramic tile and sealed concrete). An independent laboratory trial with ceramic tile inoculated with L. monocytogenes confirmed the effectiveness of the DNAH method at the 24 h time point. Overall, sensitivity of the DNAH method at 24 h relative to that of the USDA-FSIS method was 152. The DNAH method exhibited extremely high specificity, with only 1 false-positive reactions overall.

A functional dnaZ (previously designated dnaH) product, known to be involved in DNA polymerization, is required for phage λ, OX174, and M13, but not T4 or T7, growth.

An Escherichia coli HF4704S mutant temperature sensitive in deoxyribonucleic acid (DNA) synthesis and different from any previously characterized mutant was isolated. The mutated gene in this strain was designated dnaH . The mutant could grow normally at 27 C but not at 43 C, and DNA synthesis continued for an hour at a decreasing rate and then ceased. After temperature shift-up, the increased amount of DNA was 40 to 50%. When the culture was incubated at 43 C for 70 min and then transferred to 27 C, DNA synthesis resumed after about 50 min, initiating synchronously at a fixed region on the bacterial chromosome. The initiation step in DNA replication sensitive to 30 μg of chloramphenicol per ml occurs synchronously before the resumption of DNA replication after the temperature shift-down, being completed about 30 min before the start of DNA replication. When the cells incubated at 27 C in the presence of 30 μg of chloramphenicol per ml after the temperature shift-down to 27 C were transferred to 43 C with simultaneous removal of the antibiotic, no resumption of DNA replication was observed. When the culture was returned to 43 C after being released from high-temperature inhibition at 30 min before the start of DNA replication, no recovery replication was observed; whereas at 20 min, the recovery of replication was observed. These results indicated that HF4704S was temperature sensitive in the initiation of DNA replication. Analysis of HF4704S, by an interrupted conjugation experiment, indicated that gene dnaH was located at about 64 min on the E. coli C linkage map. In E. coli S1814 (a K-12 derivative), which was a dnaH ts transductant from HF4704S (C strain) with phage P1, the mutated gene ( dnaH ) was demonstrated to be closely linked to the thyA marker by conjugation and P1 transduction experiments and to be distinct from genes dnaA through dnaG .

The dnaH locus is the fourth gene to be identified as required for deoxyribonucleic acid polymerization in Escherichia coli . A temperature-sensitive mutant defective in this gene exhibited an abrupt decrease in rate of deoxyribonucleic acid synthesis when shifted to 42 C. The locus mapped in the proC-purE region of the chromosome by conjugation and was co-transducible with purE. dnaH + is carried on the F′ 13 episome and is dominant over the dnaH − mutation.

The DNA synthesis of bacteriophage phiX174 in Escherichia coli HF470S, a mutant temperature sensitive in the initiation of DNA replication (dnaHts), has been examined. In HF4704S cells, phiX174 can grow normally at 27 degree C whereas the phage cannot grow after the cessation of DNA synthesis of the host cells at 42 degrees C. Upon infection, phiX174 DNA can be injected into the host cell and the parental replicative form can be formed, but the progency replicative form cannot be synthesized at 43 degrees C in the absence of host DNA synthesis. The progency replicative form cannot be synthesized at 27 degrees C in the presence of 30 mug chloramphenicol/ml in the host cell which has been incubated for 74 min at 43 degrees C followed by transfer to 27 degrees C in the presence of 30 mug chloramphenicol/ml. When 30 mug chloramphenicol/ml is added later than 5 min after the temperature shift-down to 27 degrees C, the progency replicative form synthesis is not inhibited. Thus, the host cell function, for which the gene dnaH is responsible, has been shown to be essential to the progency replicative form production.

The dnaH mutant strain HF4704S, isolated by Sakai et al. (1974), was examined for its effect on phiX174 deoxyribonucleic acid (DNA) synthesis. It was found to carry two mutations affecting DNA synthesis. One mutation had no affect on phiX174 DNA synthesis, but did affect the ability of the mutant cells to form colonies on agar medium at 41 degrees C, and caused host DNA synthesis to cease after 1 h at 41 degrees C. The mutant marker cotransduced with ilvD at a frequency of about 9%. It seems likely that this mutation is in the dnaA gene. The second mutation affected the ability of the mutant cells to form colonies on agar medium supplemented with only 2 mug of thymine per ml, and affected both host and phiX174 DNA synthesis in medium supplemented with only 2 mug of thymine per ml. Both effects could be overcone by adding excess exogenous thymine. We were not able to unambiguously determine the map position of this mutant locus. Our data show that the DNA synthesis phenotype of the mutant strain HE4704S is governed by both these mutations, neither of which directly affects the replication of phiX174 DNA.