Increased UV-inducibility of SOS functions in a dam-3 mutant of Escherichia coli K12 uvrA.

The dam-3 mutation caused a 2--4 fold increase in the susceptibility of E. coli K-12 uvrA to UV induction of prophage lambda, induced reactivation and mutagenesis of lambda, and mutation to histidine prototrophy. The increased inducibility exceeded the level expected by UV and dam-3 acting additively and independently, and suggests that the effects of UV and dam-3 interact in some way to potentiate induction of SOS functions.     

Induction of the SOS response by hydroxyurea in Escherichia coli K12

Hydroxyurea at concentrations higher than 10(-2) M induced the recA and sfiA genes of E. coli as well as the lambda prophage by a pathway independent of the recBC genes. In addition, the hydroxyurea-mediated induction of the SOS response is accompanied by a recA-dependent decrease on the cellular ATP pool. The presence of the multicopy plasmid pPS2, harboring the nrdAB genes (encoding the ribonucleoside reductase enzyme), abolished the hydroxyurea-induced expression of the recA gene. These data lead us to suggest that induction of the SOS response by hydroxyurea is due to the blocking of DNA replication by the inhibition of the ribonucleoside reductase complex activity.     

N-acetylmuramoyl-L-alanine amidase of Escherichia coli K12. Possible physiological functions

Various experiments were carried out in an attempt to determine the possible physiological function of the N-acetylmuramoyl-L-alanine amidase purified from Escherichia coli K12 on the basis of its activity on N-acetylmuramoyl-L-alanyl-D-gamma-glutamyl-meso-diaminopimelic acid [MurNAc-LAla-DGlu(msA2pm)]. A Km value of 0.04 mM was determined with this substrate. Specificity studies revealed that compounds with a MurNAc-LAla linkage are the most probable substrates of this enzyme in vivo. Purified amidase had no effect on purified peptidoglycan and only low levels (1-2.5%) of cleaved MurNAc-LAla linkages were detected in peptidoglycan isolated from normally growing cells. However, the action of the amidase in vivo on peptidoglycan was clearly detectable during autolysis. The amidase activity of cells treated by osmotic shock, ether or toluene, as well as that of mutants with altered outer membrane composition was investigated. Attempts to reveal a transfer reaction catalysed by amidase were unsuccessful. Furthermore, by its location and specificity, amidase was clearly not involved in the formation of UDP-MurNAc. The possibility that it might be functioning in vivo as a hydrolase degrading exogeneous peptidoglycan fragments in the periplasma was substantiated by the fact that MurNAc itself and MurNAc-peptides could sustain growth of E. coli as sole carbon and nitrogen sources. Finally, out of 200 thermosensitive mutants examined for altered amidase activity, only two strains had less than 50% of the normal level of activity, whereas ten strains were found to possess more than 50%. In fact, two of the overproducers encountered presented a 4-5-fold increase in activity.     

Mutation affecting resistance of Escherichia coli K12 to nalidixic acid

A new mutation, nalD, determining resistance of Escherichia coli to nalidixic acid (NAL) is reported. The nalD mutant described is resistant to NAL at 37 degrees C but sensitive at 30 degrees C. It is defective in penetration of NAL and glycerol through the outer membrane at 37 degrees C. The nalD mutation is located half-way between 89 and 89.5 min on the E. coli genetic map.     

Survival of Escherichia coli K12 in seawater

Abstract The fate of an auxotrophic Escherichia coli K12 strain (NF1830) in coastal water was investigated. The E. coli K12 were enumerated after incubation for varying times in seawater. Incubated in raw seawater at 15 and 20°C, the NF1830 decreased from 10⁶ cfu/ml to below detection within six days of incubation, but when incubated at 7°C it persisted longer. The NF1830 was capable of cell division in sterile seawater. Growth was also shown to occur in raw seawater in the presence of autoclaved sediment. The E. coli K12 decreased in number at a much lower rate when incubated in seawater treated with eukaryotic inhibitors. These findings suggest that the die-off of the auxotrophic E. coli K12 strain seen in the raw seawater was caused by grazing of bacterial predators in the seawater.     

Vitamin para-aminobenzoic acid inhibits development of SOS function in tif-1 mutants of Escherichia coli at nonpermissive temperatures

The development of "SOS" inducible functions in lysogenic and non-lysogenic strains of Escherichia coli tif-1 sfiA11 (lambda) at nonpermissive temperature of 42 degrees C was strongly suppressed by para-aminobenzoic acid (PABA). The rate of prophage lambda induction decreased 400 times, as compared to the control level; the efficiency of W-reactivation of UV-irradiated phage lambda decreased 37.5 to 16%. PABA also inhibited to some extent (1.5 times) the process of inducible recombination on the RecF pathway. The processes of spontaneous lambda induction and W-reactivation, as well as spontaneous recombination on RecBC and RecF pathways, were not influenced by PABA. The above data are in accordance with previous studies of PABA action when the manifestation of "SOS" functions was induced by chemical mutagens. The action of PABA has been tentatively interpreted on the basis of negative control of "SOS" repair pathway.     

Defective growth functions in mutants of Escherichia coli K12 lacking a major outer membrane protein.

Various properties of mutants of Escherichia coli K12 lacking specific outer membrane proteins have been studied. ompA mutants are shown to grow less well than their parent strains under a variety of growth conditions, and after completion of growth to enter a decline phase in which viability is lost and the cells become heavily piliated and clump. They are defective in the uptake of amino acids, whereas the uptakes of the larger transport substrates ferrienterochelin and cyanocobalamin (vitamin B12) are normal. These ompA mutants also grow poorly at 42 °C. The implications of these results are discussed in terms of the function of the ompA. gene product. No growth or uptake defects were observed for ompB or tsx mutants.     

Proline excretion by Escherichia coli K12

Proline excretion from proline overproducing strains of E. coli K12 has been studied as a model chemical production system. We have isolated proline overproducing mutants of E. coli and have shown that uncontrolled synthesis is not sufficient to cause excretion of this amino acid. An episomal mutation causing proline over production has been introduced into a series of otherwise isogenic strains that bear well defined, chromosomal lesions affecting the active uptake and catabolism of L-proline. A syntropism test reveals that L-proline is excreted by overproducing strains only if transport and/or catabolism are impaired. Dansyl derivatization and chromatographic analysis of culture supernatants shows that proline is the only amino acid excreted. Batch cultures of an excreting strain in an amino acid production medium yield culture supernatants containing 1 g proline/L, whereas no proline is detectable in supernatants derived from cultures of an overproducing strain with normal transport and catabolic activities. These data reveal that genetic lesions eliminating active uptake can be used to specifically enhance metabolite excretion.     

SOS induction of the gene sulA is partly inhibited in Escherichia coli K12 cells overproducing the RecA protein

A study was made of the SOS induction of the gene sulA of Escherichia coli K12 in relation to the gene dosage of the gene recA. In experiments the sulA::lacZ fusion strain PQ37 and derivatives of PQ37 with the multi-copy plasmids pDR1453 or pBR322 were used. The SOS response was induced with nitrofurantoin, SOS induction of the gene sulA was determined on the basis of the amount of beta-galactosidase synthesized, i.e. by the SOS chromotest (Quillardet et al., 1982a). It was found in this work that cells with the plasmid pDR1453, which contain the gene recA of E. coli K12 (Sancar and Rupp, 1979), have a decreased SOS induction of the gene sulA. Cells with the plasmid pBR322 do not exhibit this decrease. Inactivation of the gene recA in the plasmid pDR1453 with preservation of the functional gene recA in the chromosome leads to a restoration of 'standard' SOS induction of the gene sulA. The results show that the amount of the gene product of the gene recA affects the SOS induction of the gene sulA.