The existence of the ksgA gene was established about forty years ago in E. coli. It was shown to be the sole methyltransferase that converts two adjacent 16S rRNA adenosines (A1518 and A1519, E. coli numbering) into N6,N6-dimethyladenosines
, modifications that appeared to hold wide phylogenetic distribution. It is now known that those modifications and the responsible methyltransferase are all but universally conserved throughout life, thus making KsgA (known as Dim1 in eukaryotes and archaea) a genetic element of the last universal common ancestor. This level of conservation, coupled with the knowledge that KsgA can be dispensed with in several bacteria, albeit with obvious growth defects
[3–8], formed the basis of a sharp paradox. If KsgA was not essential, why was it universally conserved? Since evolution is not sentimental, the cellular importance of KsgA and Dim1 was certain but remained to be discovered. In time the stated paradox has partially unraveled. In Saccharomyces cerevisiae (and most likely other eukaryotic organisms) Dim1 is an important member of the rRNA processome, and loss of Dim1 leads to the accumulation of aberrant rRNA species at the expense of functional ribosomes
. In E. coli, KsgA serves as a gate-keeper to prevent improperly assembled pre-30S subunits from entering the translation cycle
. Under normal conditions, KsgA only provides modest benefit to 30S maturation and function. However, KsgA’s importance becomes clear under stress conditions, such as growth at cold temperature.
In this work, we sought to define the importance of KsgA to the survivability of the human pathogen S. aureus and to compare our results to those in the model organism E. coli. Somewhat surprisingly, we found that S. aureus has a lesser reliance on KsgA under the conditions tested. In E. coli, overexpression of KsgA rescued the cold-sensitive phenotype of ΔksgA cells at low temperature but was deleterious for cell growth at 37°C in both knockout and parental cells. Overexpression of a catalytically inactive mutant of KsgA, E66A, was deleterious in both strains at both temperatures, even in the presence of endogenous WT protein
. We showed that in S. aureus the ksgA knock-out strain displayed a slow growth phenotype at low temperature when compared to the parental strain, similar to results in E. coli. However, unlike in E. coli, catalytic inactivation of KsgA’s enzymatic function has only mild phenotypic effects, and these effects are not dominant in the presence of WT KsgA. It is noteworthy that the negative growth effect was seen at 37°C but not at 25°C. This result was unexpected, both because ksgA knockout led to cold sensitivity and because negative effects in E. coli were exacerbated at low temperature; however, it is possible that growth at the lower temperature results in lower expression of the mutant protein and therefore a smaller negative effect.
In S. aureus, KsgA also appears to be less critical for the assembly of mature ribosomes. Experiments in E. coli showed that loss or inactivation of KsgA had obvious effects on ribosome biogenesis even under conditions where a growth phenotype was not apparent
. In other words, ribosome biogenesis is sensitive to disruptions in KsgA function that don’t affect overall cell growth. We did not see this effect in S. aureus; knockout or inactivation of KsgA resulted in, at most, slight disruption of polysome profiles even under conditions where cell growth was slowed.
On the basis of the data presented here, it would appear that in S. aureus KsgA holds less promise as a drug target than in E. coli. However, we did observe that knockout of ksgA rendered S. aureus marginally more sensitive to clinically used aminoglycoside antibiotics, similar to results seen in E. coli. A1518 and A1519 are located distal to the aminoglycoside binding site on the small ribosomal subunit; we therefore hypothesize that effects on antibiotic sensitivity are indirect, likely caused either by conformational or dynamic changes that are propagated from the site of KsgA methylation to the aminoglycoside binding site. This experiment highlights an additional difference between E. coli and S. aureus ribosomes. While lack of methylation by KsgA leads to increased sensitivity to the 4,6 class of aminoglycosides in both organisms, we see opposite effects on 4,5 aminoglycoside sensitivity. Both the KsgA target site and the aminoglycoside binding site are among the most highly conserved rRNA sequences; it is thus intriguing that distinct effects are seen between the two organisms.
Although ribosome biogenesis has not been well-studied outside of the model organisms E. coli and, to a much lesser extent, B. subtilis, it is possible that reported differences in ribosome biogenesis between Gram-negative and Gram-positive organisms are representative of an evolutionary divergence between the two groups of bacteria. One such difference is the case of the ribonuclease RNase III. RNase III is an endonuclease that is involved in processing of the pre-rRNA transcript in both E. coli and B. subtilis. However, this enzyme is strictly essential in B. subtilis but not in E. coli. Additionally, inactivation of RNase III has different effects on the maturation of 16S rRNA in the two organisms
. Further work is required to demonstrate whether these results are more broadly applicable in other bacterial species. Our work suggests differences in ribosome biogenesis between E. coli and S. aureus; it remains to be seen if the differing reliance on KsgA can be defined by a phylogenetic Gram-positive/Gram-negative split.
KsgA plays a key role in ribosome biogenesis in E. coli, which cannot be separated from its methyltransferase function
. Further evidence of KsgA’s significance in Gram-negative organisms comes from virulence studies in pathogenic organisms. Disruption of ksgA in Y. pseudotuberculosis confers an attenuated virulence phenotype on the knockout strain
, and this attenuated strain confers protection against subsequent challenge with the wild-type strain
. Additionally, mutation of ksgA in the plant pathogen E. amylovora decreases virulence
 and disruption of KsgA in S. Enteriditis reduces invasiveness
. These studies affirm that KsgA may be a novel drug target in Gram-negative organisms.
Studies on KsgA’s role in virulence have not been done in Gram-positive organisms, although in addition to the modest growth defects seen in the S. aureus ΔksgA strain disruption of the ksgA gene in the Gram-negative Mycobacterium tuberculosis was shown to negatively affect bacterial growth on solid media
. It should be noted that disruption of ksgA in Y. pseudotuberculosis produced only a slight growth defect and allowed the bacteria to survive in infected mice, even though the strain was not as virulent as the wild-type strain
. Likewise, E. amylovora mutants showed reduced virulence despite only small growth defects in vitro and the ability to grow in infected tissue
. Further studies will be required to show whether KsgA is similarly correlated with virulence in Gram-positive organisms.