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The antagonistic interaction between a potential fish probiont, Pseudomonas fluorescens strain AH2, and its target organism, Vibrio anguillarum, was investigated by studying the genetic response of the target organism when it was exposed to the antagonist. We compared the differential display of arbitrarily PCR-amplified gene transcripts in V. anguillarum serotype O1 when it was exposed to AH2 supernatant with the display of transcripts in nonexposed control cultures. Growth of V. anguillarum was immediately arrested when the organism was exposed to 50% (vol/vol) AH2 supernatant. A total of 10 potentially differentially expressed transcripts were identified. Among these we identified a gene homologous to rpoS that was induced in a dose-dependent manner when V. anguillarum was cultured in media supplemented with sterile filtered supernatant from AH2. rpoS was also induced when growth was arrested with the iron chelator 2,2-dipyridyl. A chromosomal transcript homologous to vibE that participates in vibriobactin synthesis in Vibrio cholerae was also upregulated during AH2 exposure. This transcript could represent a functionally active gene in V. anguillarum involved in biosynthesis of anguibactin or another V. anguillarum siderophore. On the pJM1 plasmid of V. anguillarum serotype O1, a pseudogene designated open reading frame E (ORF E) that contains a frameshift mutation was previously identified. The gene homologous to vibE identified in this study, interestingly, also has significant homology to ORF E on the amino acid level and does not possess the frameshift mutation. Thus, the chromosomally encoded vibE homologue could fulfil the role of the inactive plasmid-encoded ORF E pseudogene. Addition of Fe(3+) to the system eliminated the growth arrest, and the genes homologous to rpoS and vibE were not induced. To our knowledge, this is the first study linking rpoS induction to iron starvation. Taken together, the results of this study suggest that a major part of the antagonistic property exhibited by strain AH2 is caused by the ability of siderophores in the supernatant to efficiently chelate iron, which results in instant iron deprivation of the pathogen V. anguillarum and complete growth arrest.

The correlation between ribosome content and growth rate found in many bacterial species has proved useful for estimating the growth activity of individual cells by quantitative in situ rRNA hybridization. However, in dynamic environments, the stability of mature ribosomal RNA causes problems in using cellular rRNA contents for direct monitoring of bacterial growth activity in situ. In a recent paper, Cangelosi and Brabant suggested monitoring the content of precursors in rRNA synthesis (pre-rRNAs) as an alternative approach. These are rapidly broken down after the cessation of bacterial growth. We have applied fluorescence in situ hybridization of pre-16S rRNA to Escherichia coil cells growing in vitro in extracts from two different compartments of the mouse intestine: the caecal mucus layer, where E. coli grew rapidly, and the contents of the caecum, which supported much slower bacterial growth. The amounts of 23S rRNA and pre-16S rRNA measured for E. coil growing in intestinal mucus corresponded to that expected for bacteria with the observed growth rate. In contrast, the slow-growing E. coli cells present in intestinal contents turned out to have an approximately ninefold higher content of pre-16S rRNA than cultures of the same strain growing rapidly in rich media. We present results suggesting that the mouse intestinal contents contain an agent that inhibits the growth of E. coli by disturbing its ability to process pre-16S rRNA.

An in situ PCR protocol by which we can monitor the presence or absence of lac mRNA in individual cells of a Salmonella typhimurium F' lac+ strain has been developed. In this protocol, fixed cells are permeabilized with lysozyme and subjected to a seminested reverse transcriptase PCR using reporter molecule-labeled primers, and subsequently, intracellular reporter molecules are detected microscopically at the individual-cell level by use of a horseradish peroxidase-conjugated antifluorescein antibody assay. In order to determine the sensitivity of the in situ PCR assay, the ability to detect lac mRNA in suboptimally isopropyl-beta-D-thiogalactopyranoside-induced cells was investigated. By use of a single-cell beta-galactosidase assay, it was confirmed that homogeneous suboptimally induced cultures of S. typhimurium F' lacY cells could be established, and the number of functional lac mRNAs in individual cells was estimated from standard population level beta-galactosidase assays. Cells estimated to contain a single lac mRNA were detected as containing lac mRNA by the in situ PCR method. Conclusively, we demonstrate the potential of in situ PCR for detection of even poorly expressed mRNA in individual bacterial cells.