Functional activity of E. coli RNase R in the Antarctic Pseudomonas syringae Lz4W

Abstract

Background: In Antarctic P. syringae RNase R play an essential role in the processing of 16S and 5S rRNA, thereby playing an important role in cold-adapted growth of the bacterium. This study is focused on deciphering the in vivo functional activity of mesophilic exoribonuclease R and its catalytic domain (RNB) in an evolutionary distant psychrophilic bacterium Pseudomonas syringae Lz4W.

Results: Our results confirm that E. coli RNase R complemented the physiological functions of the psychrophilic bacterium P. syringae RNase R and rescued the cold-sensitive phenotype of Pseudomonas syringae ∆rnr mutant. More importantly, the catalytic domain (RNB) of the E. coli RNase R is also capable of alleviating the cold-sensitive growth defects of ∆rnr mutant as seen with the catalytic domain (RNB) of the P. syringae enzyme. The Catalytic domain of E. coli RNase R was less efficient than the Catalytic domain of P. syringae RNase R in rescuing the cold-sensitive growth of ∆rnr mutant at 4°C, as the ∆rnr expressing the RNB^Ec (catalytic domain of E. coli RNase R) displayed longer lag phase than the RNB^Ps (Catalytic domain of P. syringae RNase R) complemented ∆rnr mutant at 4°C. Altogether it appears that the E. coli RNase R and P. syringae RNase R are functionally exchangeable for the growth requirements of P. syringae at low temperature (4°C). Our results also confirm that in P. syringae the requirement of RNase R for supporting the growth at 4°C is independent of the degradosomal complex.

Conclusion: E. coli RNase R (RNase R^Ec) rescues the cold-sensitive phenotype of the P. syringae Δrnr mutant. Similarly, the catalytic domain of E. coli RNase R (RNB^Ec) is also capable of supporting the growth of Δrnr mutant at low temperatures. These findings have a vast scope in the design and development of low-temperature-based expression systems.

Keywords: Catalytic domain; Cold-adapted enzymes; Degradosome; Exoribonuclease R; Functional complementation; Psychrophiles; RNA processing.

Fig. 1

Domain organization of RNase R. Schematic representing the domain organization in E.coli and P. syringae RNase R. Individual domains are shaded in separate colors and numbers represent the amino acids in the primary sequence of protein

Fig. 2

Expression of E.coli RNase R. Expression of RNase RPs and RNase REc in ∆rnr strain was analyzed by western blotting where cell lysate from ∆rnr strains expressing RNase RPs and RNase REc were transferred to a nylon membrane and probed by anti-His antibodies

Fig. 3

Expression of RNB (Catalytic) domain of E. coli RNase R.  (a) Color-coded schematic diagram showing different domains (color shaded) and position of primers employed for amplification of catalytic RNB domain. The numbers represent the nucleotide base pairs in rnr gene. (b) Expression of RNB domain in ∆rnr strain complemented by pGLRNBEc was analyzed by western blotting. Cell lysate from wild-type cells (Lane 2), ∆rnrpGLRNBEc strain (Lane 1) were loaded on polyacrylamide gel, transferred to a nylon membrane, and probed with anti RNase RPs antibodies

Fig. 4

Multiple sequence alignment [T-coffee, www.ebi.ac.uk] of the amino acid sequence of the RNA helicases. Accordingly, B. subtilis has been indicated as B whereas E. coli has been abbreviated as E. Similarly P. syringae Lz4W, P. aeruginosa, and P. florescens have been indicated as Lz4W, P_1, and P_2 respectively. The alignment results also illustrate the identity among amino acid residues in different [N and C] regions of the protein

Fig. 5

Mesophilic RNase R complements cold-sensitive phenotype of ∆rnr. (a) Growth profile of wild-type, ∆rnr, ∆rnrpGLrnrPs, and ∆rnrpGLrnrEc strains at 22°C and (b) at 4°C confirmed over-expression of RNase R^Ec  from broad host range plasmid (pGL10) complements cold-sensitive phenotype of Pseudomonas syringae ∆rnr strain. For measurement of growth, samples were collected from each culture at regular intervals, OD at 600 nm was recorded and plotted against time. Each growth curve was performed at least three times

Fig. 6

Complementation of ∆rnr strain by catalytic domain (RNB) of E. coli RNase R. (a) Growth analysis of P. syringae wild type, ∆rnr, ∆rnrpGLRNB^Ps, and ∆rnrpGLRNB^Ec strains at 22°C and (b) at 4°C established that complementation of cold-sensitive ∆rnr strain by RNB^Ec alleviates the cold-sensitive phenotype of mutant strain but with a long Lag phase even longer than with RNB^Ps. For measurement of growth in cell cultures at 22°C or 4°C, samples were collected from each culture at regular intervals, and their OD at 600 nm was recorded and plotted against time. Each growth curve was repeated at least three times