Aggregatibacter actinomycetemcomitans serotype f O-polysaccharide mediates coaggregation with Fusobacterium nucleatum.

BACKGROUND/AIMS: Intergeneric bacterial coaggregation may play an important role in plaque development. METHODS: In this study we investigated the coaggregation reaction between two periodontal pathogens, Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum. RESULTS: Previous studies showed that A. actinomycetemcomitans serotype b strains coaggregate with F. nucleatum strain PK1594, and that A. actinomycetemcomitans serotype b O-polysaccharide (O-PS) is the receptor responsible for coaggregation between A. actinomycetemcomitans and F. nucleatum. A. actinomycetemcomitans serotype f O-PS has been shown to be structurally and antigenically related to serotype b O-PS. In the present study we show that A. actinomycetemcomitans strain CU1060N, a serotype f strain, also coaggregated with F. nucleatum PK1594. Like coaggregation between serotype b strains and F. nucleatum, coaggregation between CU1060N and F. nucleatum was inhibited by galactose. An O-PS mutant of CU1060N failed to coaggregate with F. nucleatum. CONCLUSION: We concluded that A. actinomycetemcomitans serotype f O-PS, like serotype b O-PS, mediates coaggregation between A. actinomycetemcomitans and fusobacteria.

Prevalence and distribution of bacteriophage phi Aa DNA in strains of Actinobacillus actinomycetemcomitans.

phi Aa is a bacteriophage that was originally isolated by induction of a lysogenic strain of the oral bacterium Actinobacillus actinomycetemcomitans. Since the discovery of phage phi Aa, additional phages infecting several other strains of A. actinomycetemcomitans have been identified. To determine the prevalence of phi Aa or phi Aa-related temperate phages in this species, a phi Aa-specific DNA probe was prepared to screen for homologous sequences among 42 strains of A. actinomycetemcomitans. Fourteen (33%) of the 42 strains examined contained DNA sequences that hybridized with the phage phi Aa probe. A bacteriophage designated phi Aa33384 was isolated by induction from one of the strains (ATCC 33384) that contained a sequence that hybridized with the phi Aa probe. The phi Aa probe hybridized with the DNA extracted from bacteriophage phi Aa33384. The distribution of the phage phi Aa sequence among A. actinomycetemcomitans serotypes was 5/13 (38%) of the serotype a strains, 0/16 (0%) of the serotype b strains, and 9/13 (69%) of the serotype c strains. The results of this investigation suggest that the target sequence prepared from the phage phi Aa genome is fairly common in the A. actinomycetemcomitans chromosome, and that the sequence is distributed among the A. actinomycetemcomitans serotypes in a seemingly nonrandom manner.

Conjugal transfer of broad-host-range incompatibility group P and Q plasmids from Escherichia coli to Actinobacillus actinomycetemcomitans.

The first example of conjugal transfer of DNA from Escherichia coli to the periodontal pathogen Actinobacillus actinomycetemcomitans is presented. Derivatives of the incompatibility group P (IncP) plasmid RK2 successfully transferred from an E. coli donor to an A. actinomycetemcomitans recipient. The resulting A. actinomycetemcomitans transconjugants transferred the plasmids back to E. coli recipients. The IncP transfer functions were also used in trans to mobilize the IncQ plasmid pBK1 from E. coli to A. actinomycetemcomitans. The IncP and IncQ plasmids both transferred into A. actinomycetemcomitans at high frequencies (0.3 to 0.5 transconjugants per donor) and showed no gross deletions, insertions, or rearrangements. Determinations of MICs of various antibiotics for the A. actinomycetemcomitans transconjugant strains demonstrated the expression of ampicillin, chloramphenicol, and kanamycin resistance determinants.

Characterization and physical mapping of the genome of bacteriophage phi Aa from Actinobacillus actinomycetemcomitans.

The size, configuration and restriction map of Actinobacillus actinomycetemcomitans bacteriophage phi Aa DNA was determined by means of restriction endonuclease analysis. Digestion of the phi Aa DNA with restriction enzymes Hind III, Eco RI and Sal I produced 6, 5, and 4 fragments, respectively. Based upon the sum of the sizes of the restriction fragments of these enzymes, the DNA was estimated to be 47.2 kilobase pairs in length. A restriction map was constructed using Hind III and Sal I. Incubation with exonuclease Bal 31 for increasing lengths of time resulted in progressive hydrolysis of the DNA, as expected for a linear molecule. No sub-molar fragments or diffuse bands were observed in the agarose gels of the restriction endonuclease digests of the phi Aa DNA. Attempts at ligating the ends of the DNA were consistently unsuccessful. Therefore, we found no evidence for cohesive ends, a circular permutation of the genome or for headful packaging mechanism from a concatameric DNA precursor.

Differentiation of lethal and nonlethal, kor-regulated functions in the kilB region of broad host-range plasmid RK2.

In broad host-range plasmid RK2, several kil loci (kilA, kilB, kilC, kilE) and the replication initiator gene (trfA) are regulated by combination of kor determinants (korA, korB, korC, korE) in a regulatory network known as the kil-kor region. Although the kil determinants are not essential for replication, their coregulation with trfA suggests an involvement in plasmid maintenance or host-range. Plasmids carrying the cloned kilB region of RK2 cannot be maintained in the absence of korB owing to two phenotypically distinguishable, kor-regulated determinants: (1) kilB1 (kilD), which can be controlled by korA or korB, and (2) kilB2, which requires korB for control. In this study, we have determined the nature of the functions responsible for the kor-sensitive phenotypes of the kilB region. We found that insertion of transcription terminators within or downstream of the trfA operon allows plasmids carrying the kilB1 portion of the kilB region to be maintained in cells lacking korA or korB. In addition, mutants of the kilB1 region that can be maintained in the absence of korA and korB have alterations in the trfA promoter. These results show that the phenotype of the cloned kilB1 region in kor-deficient cells depends on trfA transcription but does not involve expression of any gene of the trfA operon. Therefore, the kilB1 determinant is not a structural gene. The phenotype results from entry of trfA-initiated transcription into adjacent sequences of the plasmid vector. The ability to block the kilB2 phenotype with transcriptional terminators allowed us to show conclusively that the kilB2 determinant is a host-lethal gene (klbA) whose regulation is dependent on korB. These findings have implications for the structure of the basic replicon of RK2.

Replication control in promiscuous plasmid RK2: kil and kor functions affect expression of the essential replication gene trfA.

We previously reported that broad-host-range plasmid RK2 encodes multiple host-lethal kil determinants (kilA, kilB1, kilB2, and kilC) which are controlled by RK2-specified kor functions (korA, korB, and korC). Here we show that kil and kor determinants have significant effects on RK2 replication control. First, korA and korB inhibit the replication of certain RK2 derivatives, unless plasmid replication is made independent of the essential RK2 gene trfA. Second, kilB1 exerts a strong effect on this interaction. If the target plasmid is defective in kilB1, sensitivity to korA and korB is enhanced at least 100-fold. Thus, korA and korB act negatively on RK2 replication, whereas kilB1 acts in a positive manner to counteract this effect. A mutant RK2 derivative, resistant to korA and korB, was found to have fused a new promoter to trfA, indicating that the targets for korA and korB are at the 5' end of the trfA gene. We constructed a trfA-lacZ fusion and found that synthesis of beta-galactosidase is inhibited by korA and korB. Thus korA, korB, and kilB1 influence RK2 replication by regulating trfA expression. We conclude that the network of kil and kor determinants is part of a replication control system for RK2.

Genetic interactions of broad host-range plasmid RK2: evidence for a complex replication regulon.

The kil and kor genes of RK2 are novel genetic determinants further that the kil and kor network constitutes a replication regulon, and that perhaps the function of this regulon is to ensure expression of trfA at appropriate levels. The complexity of this regulon may reflect an ability of the system to adapt to the intracellular environments of a variety of hosts. Indeed, there is tantalizing evidence that regions encoding kil or kor genes are important to host range (1,2,6,28; Schmidhauser and Helinski, pers. comm.). We are therefore hopeful that the study of these genes and the eventual determination of the molecular basis of their actions will lead to a complete understanding of the replication control and broad host range capability of IncP plasmids.

The relationship of the lithium erythrocyte: plasma ratio to plasma lithium level.

The relationship of the lithium erythrocyte:plasma ratio to plasma lithium concentration was reviewed in inpatients and outpatients with affective disorders. For some patients, there was a linear correlation between the erythrocyte lithium:plasma lithium ratio and the plasma lithium concentration. For these patients a graph of the slopes and intercepts of the lithium erythrocyte:plasma ratio vs. plasma lithium data formed a line that was not significantly different from the data of Lee et al. (1975). Significant correlations were found between the slopes and intercepts of the lithium erythrocyte:plasma ratio vs. plasma lithium data and the magnitude of active lithium efflux (Ko) from the erythrocyte. Our data confirm the finding of Lee et al. (1975) that the lithium erythrocyte:plasma ratio is dependent on the plasma lithium concentration. We relate this finding to lithium efflux from the erythrocyte.

Plasma and erythrocyte magnesium levels in patients with primary affective disorder during chronic lithium treatment.

Plasma and erythrocyte magnesium concentrations were measured in 79 clinic outpatients who had histories of bipolar or unipolar primary affective disorder and who were being chronically treated with lithium carbonate (60 patients) or placebo (19 patients). Although slight differences in the mean concentration of magnesium in plasma and erythrocytes were noted with lithium treatment, diagnosis, and sex, the differences overall failed to achieve statistical significance. In contrast to prior reports demonstrating increases in plasma magnesium during acute lithium carbonate treatment of affectively ill patients, these data suggest that the pre-lithium steady state is achieved during chronic lithium treatment.