Prediction of CsrA-regulating small RNAs in bacteria and their experimental verification in Vibrio fischeri

Abstract

The role of small RNAs as critical components of global regulatory networks has been highlighted by several recent studies. An important class of such small RNAs is represented by CsrB and CsrC of Escherichia coli, which control the activity of the global regulator CsrA. Given the critical role played by CsrA in several bacterial species, an important problem is the identification of CsrA-regulating small RNAs. In this paper, we develop a computer program (CSRNA_FIND) designed to locate potential CsrA-regulating small RNAs in bacteria. Using CSRNA_FIND to search the genomes of bacteria having homologs of CsrA, we identify all the experimentally known CsrA-regulating small RNAs and also make predictions for several novel small RNAs. We have verified experimentally our predictions for two CsrA-regulating small RNAs in Vibrio fischeri. As more genomes are sequenced, CSRNA_FIND can be used to locate the corresponding small RNAs that regulate CsrA homologs. This work thus opens up several avenues of research in understanding the mode of CsrA regulation through small RNAs in bacteria.

Figure 1

Distribution of AGGA/ARGGA-binding motifs in intergenic regions. (A) Frequency distribution [f(N_m)] of the maximal number (N_m) of AGGA/ARGGA-binding motifs in intergenic regions of E.coli using a sliding window covering 240 bp. Two intergenic regions are clearly separated from the genomic background. Closed bars indicate the top strand and open bars indicate the bottom strand. (B) The same as (A) but for V.fischeri.

Figure 2

Secondary structure of CsrB1 in V.fischeri. Predicted secondary structure [obtained using MFOLD (31)] for CsrB1 in V.fischeri showing multiple AGGA/ARGGA sequence motifs in the loop regions. The secondary structure for CsrB2 is almost identical to that of CsrB1 since the two sRNAs are highly homologous.

Figure 3

Sequence logos for upstream binding sites of predicted sRNAs. The sequence logos [generated using Web Logo (40)] for conserved upstream sites for all the known and predicted (A) csrB, (B) rsmX/Y/Z and (C) csrC sRNA genes.

Figure 4

Transcription of csrB1 and csrB2. (A) β-Galactosidase activity levels of recombinant DH5α strains encoding csrB1- or csrB2-lacZ transcriptional fusions in pSP417. Background levels of β-galactosidase produced from the negative control pSP417 were 0.063 ± 0.004 RLU. Error bars represent the standard deviation of assays performed in triplicate from three independent samples. (B) Northern blot analysis of the rate of transcription of csrB1 and csrB2 in V.fischeri ES114 grown to different OD values as indicated using csrB2 sequences as a probe. Identical results were obtained when csrB1 sequences were used as a probe (data not shown). The blot shown is representative of two independent experiments. The migration of RNA size standards is indicated on the right.

Figure 5

Effects of V.fischeri proteins on glycogen regulation. Recombinant E.coli MG1655 overexpressing V.fischeri CsrA, CsrB1, CsrB2 or no protein from V.fischeri were grown on Kornberg agar plates supplemented with 1 mM IPTG and 100 μg/ml ampicillin and qualitatively assayed for levels of glycogen production.