Jonathan T. Ou

Nucleotide and Amino Acid Sequences of oriT-traM-traJ-traY-traA-traL Regions and Mobilization of Virulence Plasmids of Salmonella enterica Serovars Enteritidis, Gallinarum-Pullorum, and Typhimurium

The virulence plasmid of Salmonella enterica serovar Gallinarum-Pullorum (pSPV) but not those of Salmonella enterica serovars Enteritidis (pSEV) and Typhimurium (pSTV) can be readily mobilized by an F or F-like conjugative plasmid. To investigate the reason for the difference, the oriT-traM-traJ-traY-traA-traL regions of the three salmonella virulence plasmids (pSVs) were cloned and their nucleotide and deduced amino acid sequences were examined. The cloned fragments were generally mobilized more readily than the corresponding full-length pSVs, but the recombinant plasmid containing the oriT of pSPV was, as expected, more readily mobilized, with up to 100-fold higher frequency than the recombinant plasmids containing the oriT of the other two pSVs. The nucleotide sequences of the oriT-traM-traJ-traY-traA-traL region of pSEV and pSTV were almost identical (only 4 bp differences), but differed from that of pSPV. Major nucleotide sequence variations were found in traJ, traY, and the Tra protein binding sites sby and sbm. sby of pSPV showed higher similarity than that of pSEV or pSTV to that of the F plasmid. The reverse was true for sbm: similarity was higher with pSEV and pSTV than with pSPV. In the deduced amino acid sequences of the five Tra proteins, major differences were found in TraY: pSEVs TraY was 75 amino acids, pSTVs was 106 amino acids, and pSPVs was 133 amino acids; and there were duplicate consensus betaalphaalpha fragments in the TraY of pSPV and F plasmid, whereas there was only a single betaalphaalpha fragment in that of pSEV and pSTV.

Role of surface exclusion genes in lethal zygosis in Escherichia coli K12 mating.

SummaryWe find that diaminopimelic acid in the recipient membrane is released into the medium during bacterial matings, indicating that membrane damage was inflicted on the recipient by the donor, probably for forming a channel for DNA transfer. When the damage is extensive, as in matings with an excess of Hfr bacteria, the F- bacteria are killed (lethal zygosis). The transfer of a large amount of DNA in Hfr matings appears to enhance the killing. In analogous F+xF- (Nalr) matings, on the other hand, killing of F- bacteria does not occur unless F plasmid transfer is inhibited by a substance like nalidixic acid. The F- bacteria are killed, suggesting that F plasmids contain genes that express immunity to lethal zygosis in the recipient. For example, bacteria containing surface exclusion-deficient mutants of F plasmids, such as traS- and traT-, induce lethal zygosis in F- bacteria and are susceptible to it. Various tra- polar mutants that abolish surface exclusion are also susceptible to lethal zygosis when mated with Hfr bacteria. Kinetic experiments indicate that in F+ (wild type) x F- matings, immunity to lethal zygosis is expressed in the F- recipient within 1/4 division time, whereas a complete expression of surface exclusion requires more than 1 division time. Thus, a complete change in all receptor sites seems to be required for the expression of surface exclusion.

Escherichia coli K-12 F− mutants that form mating aggregates but form transconjugants with low frequencies

SummaryWe have isolated Escherichia coli F− mutants which, when mated with either Hfr or F′, can form stable mating aggregates well but produce transconjugants with reduced frequencies. Selection procedure and other tests rule out the possibility that they are Rec− strains. These mutants can be classified into two types: type I mutants can induce conjugal DNA replication in the donor, yet form transconjugants poorly; whereas, type II mutants induce conjugal DNA replication with poor efficiencies in the donor. Further tests indicate that type I mutants are very sensitive to lethal zygosis and their membranes, both inner and outer, show alterations in protein composition, whereas type II mutants are insensitive to lethal zygosis, and have an obvious alteration in the protein composition of their outer membrane. These results suggest that type I is defective in transconjugant formation primarily due to a change in the inner membrane, whereas type II is defective in generating a mating signal, which induces donor conjugal DNA replication, due to an alteration in the outer membrane.

Mating due to loss of surface exclusion as a cause for thermosensitive growth of bacteria containing the Rtsl plasmid

SummaryAt 25° C, Rtsl+ bacteria grow to about 5x109 bacterial/ml before leveling off, whereas at 42° C they grow from 2.6x108 bacteria/ml for only 2.3 generations after temperature shift before the growth is inhibited with a zig-zag pattern at the plateau. When diluted, Rtsl+ bacteria grow rapidly at 42° C, until the concentration reaches as high as the undiluted 42° C culture when growth measured by colony counts stops and the zig-zag pattern again appears. This density-dependent growth inhibition is not due to the presence of stable growth inhibitor(s). Mating experiments show that at 42° C, Rtsl+ bacteria retain good donor ability; at the same time, they become good recipients in mating with Hfr (Rtsl) bacteria. SDS-PAGE reveals that membranes are altered at 42° C. Examination of DNA synthesis indicates that chromosomal DNA is synthesized at both 25° C and 42° C at high bacterial concentration, but that of the Rtsl plasmid is slowed down at 42° C. The labeling experiments suggest that in 2 h there are 2 rounds of plasmid replication at 25° C, 3.5 rounds at 42° C when bacteria are diluted, and 0.6 rounds at 42° C when bacteria are not diluted. These results suggest that the growth inhibition of Rtsl+ bacteria at 42° C is probably the consequence of mating initiated due to loss of surface exclusion.