Expression of the pspABCDE operon of Escherichia coli is induced upon infection by filamentous phage and by many other stress conditions, including defects in protein export. Expression of the operon requires the alternative sigma factor 54 and the transcriptional activator PspF. In addition, PspA plays a negative regulatory role, and the integral-membrane proteins PspB and PspC play a positive one. In this study, we investigated whether the suggested protein-protein interactions implicated in this complex regulatory network can indeed be demonstrated. Antisera were raised against PspB, PspC, and PspD, which revealed, in Western blotting experiments, that PspC forms stable sodium dodecyl sulfate-resistant dimers and that the hypothetical pspD gene is indeed expressed in vivo. Fractionation experiments showed that PspD localizes as a peripherally bound inner membrane protein. Cross-linking studies with intact cells revealed specific interactions of PspA with PspB and PspC, but not with PspD. Furthermore, affinity-chromatography suggested that PspB could bind PspA only in the presence of PspC. These data indicate that regulation of the psp operon is mediated via protein-protein interactions.

Although their exact function remains enigmatic, bifidobacteria are among the first colonizers of the newborn infant gut and further develop into abundant communities, notably in response to diet. Therefore, the transcriptional responses of bifidobacteria in rapidly processed fecal samples from young infants that were fed either breast milk or a formula containing a mixture of galacto-and fructo-oligosaccharides were studied. The presence and diversity of the bifidobacterial fecal communities were determined using PCR-denaturing gradient gel electrophoresis and quantitative real-time PCR for specific species. Changes in the total number of bifidobacteria as well as in species diversity were observed, indicating the metabolic activities of the bifidobacteria within the infant gut. In addition, total RNAs isolated from infant feces were labeled and hybridized to a bifidobacterium-specific microarray comprising approximately 6,000 clones of the major bifidobacterial species of the human gut. Approximately 270 clones that showed the most prominent hybridization with the samples were sequenced. Fewer than 10% of the hybridizing clones contained rRNA genes, whereas the vast majority of the inserts showed matches with protein-encoding genes predicted to originate from bifidobacteria. Although a wide range of functional groups was covered by the obtained sequences, the largest fraction (14%) of the transcribed genes assigned to a functional category were predicted to be involved in carbohydrate metabolism, while some were also implicated in exopolysaccharide production or folate production. A total of three of the above-described protein-encoding genes were selected for quantitative PCR and sequence analyses, which confirmed the expression of the corresponding genes and the expected nucleotide sequences. In conclusion, the results of this study show the feasibility of obtaining insight into the transcriptional responses of intestinal bifidobacteria by analyzing fecal RNA and highlight the in vivo expression of bifidobacterial genes implicated in host-related functions.

SummaryA bifidobacterial mixed‐species microarray platform was used in a proof‐of‐principle study to address the composition and development of bifidobacteria in DNA extracted from faecal samples. These were collected in a time‐course of 2 years since birth and derived from human infants that were breastfed, standard formula‐fed or received a prebiotic formula during their weaning period. A set of over 50 samples was analysed, testifying for the throughput of the designed platform for multiple genome hybridizations. The generated data revealed that faecal samples of breastfed infants contained a high abundance of genomic DNA homologous to Bifidobacterium breve. In contrast, faecal samples from standard formula‐fed infants lacked detectable amounts of this B. breve DNA but contained genes with high similarity to B. longum. Remarkably, infants that received breastmilk and later a prebiotic formula consisting of a standard formula milk containing a mixture of specific galacto‐ and fructo‐oligosaccharides, continued to harbour a B. breve‐dominant faecal population. One infant that received standard formula in combination with the additional B. lactis Bb12 culture, contained significant amounts of faecal DNA belonging to Bb12 but only during the period of ingestion. The microarray platform showed sufficient sensitivity to analyse the B. breve group at the strain level. Overall, the B. breve populations observed in the faecal samples of the studied infants showed a stable composition over time and were unique per infant. In conclusion, our results show the applicability of comparative genome hybridization to study bifidobacterial populations in infant faecal samples without the use of any amplification step.

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