Expression profiling reveals Spot 42 small RNA as a key regulator in the central metabolism of Aliivibrio salmonicida

Abstract

Background: Spot 42 was discovered in Escherichia coli nearly 40 years ago as an abundant, small and unstable RNA. Its biological role has remained obscure until recently, and is today implicated in having broader roles in the central and secondary metabolism. Spot 42 is encoded by the spf gene. The gene is ubiquitous in the Vibrionaceae family of gamma-proteobacteria. One member of this family, Aliivibrio salmonicida, causes cold-water vibriosis in farmed Atlantic salmon. Its genome encodes Spot 42 with 84% identity to E. coli Spot 42.

Results: We generated a A. salmonicida spf deletion mutant. We then used microarray and Northern blot analyses to monitor global effects on the transcriptome in order to provide insights into the biological roles of Spot 42 in this bacterium. In the presence of glucose, we found a surprisingly large number of ≥ 2X differentially expressed genes, and several major cellular processes were affected. A gene encoding a pirin-like protein showed an on/off expression pattern in the presence/absence of Spot 42, which suggests that Spot 42 plays a key regulatory role in the central metabolism by regulating the switch between fermentation and respiration. Interestingly, we discovered an sRNA named VSsrna24, which is encoded immediately downstream of spf. This new sRNA has an expression pattern opposite to that of Spot 42, and its expression is repressed by glucose.

Conclusions: We hypothesize that Spot 42 plays a key role in the central metabolism, in part by regulating the pyruvat dehydrogenase enzyme complex via pirin.

Figure 1

Synteny comparison and sequence alignment of spf and VSsrna24. (A) The order of genes that flank spf and other sRNA genes (filled arrows) in A. salmonicida and E. coli are shown. Question marks indicate genes with unknown function. (B) Sequence alignment of spf and VSsrna24 from selected members of Vibrionaceae, E. coli and Serratia marcescens. The CRP binding site of E. coli, and -10 and -35 promoter regions in front of spf and VSsrna24 are based on knowledge from E. coli [7,8]. The vertical open arrows denote the 5' end of Spot 42 and VSsrna24 based on 5' RACE data on A. salmonicida presented in this work. The horizontal arrows (#1-4) denote areas deleted in the spf (#1-2) and VSsrna24 (#3-4) deletion strains. Numbers in parenthesizes denote numbers of nt to the nearest CDS. Asterisks indicate invariable positions.

Figure 2

Monitoring Spot 42 and VSsrna24 expression under different treatments. (A) A. salmonicida was cultured for approximately 30 hours (h) starting at OD_{600 nm} 0.1, and ending at 1.8 (stationary phase). A typical growth trajectory of A. salmonicida in LB is illustrated in the figure. Samples were collected throughout the growth cycle. The culture was split and 1 mM cAMP was added at OD_{600 nm} 0.4 to one half. Similarly, a second culture was split and 5 mM glucose was added at OD_{600 nm} 1.2. Untreated cells were used as control. (B) Samples collected in A were subjected to Northern blot analysis. From the control culture samples were collected at OD_{600 nm} 0.2-1.7, and after the additions of cAMP or glucose samples were collected after 5-240 min (OD_{600 nm} 0.4-0.8) or 5-360 (OD_{600 nm} 1.2-1.5) min, respectively. Radio-labeled double-stranded DNA probes were used to monitor the levels of Spot 42 and VSsrna24, and 5S rRNA was used to normalize the result. Fold change values ≥ 2 are shown below samples, and are always comparisons between treated and untreated control samples, at same ODs.

Figure 3

Overview of microarray results with spf deletion mutant compared to the wild-type strain. Bacteria were grown to OD_{600 nm} 0.4, then the culture was split and 44.4 mM glucose was added to one half. Cells were collected after 15 min. CDSs with differential gene expression corresponding to up- or downregulation above or below 1.5 fold are divided into functional categories as defined by the Sanger Institute Pathogen Sequencing Unit http://genprotec.mbl.edu/files/MultiFun.html. Numbers in parenthesises indicate in percentage the share of the total number of genes in the genome that each class represents.

Figure 4

Validation of selected microarray results with Northern blot analysis. (A) RNAs from wild-type and spf and VSsrna24 deletion mutants (Δspf and ΔVSsrna24, respectively) were separated on 5% denaturating polyacrylamide gels, transferred to membranes and tested for presence of the sRNAs Spot 42, RyhB, Qrr and VSsrna24. 5S rRNA was used as control and to normalize the results. Plus (+) indicates that glucose was added to the culture 15 min prior to sampling, whereas minus indicates that no glucose was added. Numbers to the right of gel pictures indicate the length of RNAs as measured from the gel. Fold change values ≥ 2 are shown below samples, and are always comparisons between deletion mutants and wild-type control samples, at same conditions. (B) Same samples as described above separated on a 1.2% denaturating formamide gel.

Figure 5

A model for Spot 42 regulation in A. salmonicida. The model is based on microarray and Northern blot analyses from this study, which suggest that Spot 42 downregulates Pirin (key regulator of puruvat dehydrogenase complex in central metabolism), genes for uptake/catabolism of less preferred sugars in a carbon catabolite repression (CRR)-like manner, and genes involved in motility and chemotaxis. The results also suggest that Spot 42 activates expression of other ncRNAs (e.g., the sRNA RyhB).