Figure 5.

Ribosomal defects due to loss of YbeY in S. meliloti. (A) Cell lysates from the exponentially growing cells were analyzed in a sucrose gradient as described in Material and methods. The S. meliloti strain lacking YbeY (dashed line) shows a reduced amount of 70S ribosomes and an increase in the 30S and 50S ribosomal particles compared to the wild type S. meliloti (Solid line). (B) Total RNA prepared from the wild type S. meliloti (SmybeY+) and S. meliloti lacking YbeY (SmΔybeY) were separated by an agarose/ Synergel electrophoresis. SmΔybeY strain accumulates increased levels of an unknown rRNA band (Red arrow) compared to the SmybeY+ strain, intermediate in size between the 2.6Kb RNA and the 16S RNA. (C) Northern Blot using 16S specific ³²P labeled probe detects the unknown band in the SmΔybeY strain carrying the empty vector (pRF771) but not in the SmΔybeY strain carrying the SmYbeY expressing plasmid (pRF-SmYbeY). Increasing concentrations of RNA (1, 2.5 and 5 μg) used from lane 1 to lane 3 and from lane 4 to lane 6. (D) Northern blot using the 23S specific probe fails to detect the unknown rRNA band supporting the conclusion that the unknown band in SmΔybeY strain is a 16S rRNA species. (E) Total RNA, prepared from the S. meliloti lacking YbeY (SmΔybeY) carrying plasmids that are empty (pRF771) or expressing wild type SmYbeY (pRF-SmYbeY) or SmYbeY R69A (pRF-SmYbeY R69A), were separated by an agarose/Synergel electrophoresis. SmΔybeY strain complemented with wild type SmYbeY did not accumulate pre-16S rRNA while complementation with SmYbeY R69A accumulated pre-16S rRNA.