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1.  Inactivation of KsgA, a 16S rRNA Methyltransferase, Causes Vigorous Emergence of Mutants with High-Level Kasugamycin Resistance ▿  
The methyltransferases RsmG and KsgA methylate the nucleotides G535 (RsmG) and A1518 and A1519 (KsgA) in 16S rRNA, and inactivation of the proteins by introducing mutations results in acquisition of low-level resistance to streptomycin and kasugamycin, respectively. In a Bacillus subtilis strain harboring a single rrn operon (rrnO), we found that spontaneous ksgA mutations conferring a modest level of resistance to kasugamycin occur at a high frequency of 10−6. More importantly, we also found that once cells acquire the ksgA mutations, they produce high-level kasugamycin resistance at an extraordinarily high frequency (100-fold greater frequency than that observed in the ksgA+ strain), a phenomenon previously reported for rsmG mutants. This was not the case for other antibiotic resistance mutations (Tspr and Rifr), indicating that the high frequency of emergence of a mutation for high-level kasugamycin resistance in the genetic background of ksgA is not due simply to increased persistence of the ksgA strain. Comparative genome sequencing showed that a mutation in the speD gene encoding S-adenosylmethionine decarboxylase is responsible for the observed high-level kasugamycin resistance. ksgA speD double mutants showed a markedly reduced level of intracellular spermidine, underlying the mechanism of high-level resistance. A growth competition assay indicated that, unlike rsmG mutation, the ksgA mutation is disadvantageous for overall growth fitness. This study clarified the similarities and differences between ksgA mutation and rsmG mutation, both of which share a common characteristic—failure to methylate the bases of 16S rRNA. Coexistence of the ksgA mutation and the rsmG mutation allowed cell viability. We propose that the ksgA mutation, together with the rsmG mutation, may provide a novel clue to uncover a still-unknown mechanism of mutation and ribosomal function.
PMCID: PMC2612157  PMID: 19001112
2.  Two Genetic Loci for Resistance to Kasugamycin in Escherichia coli 
Journal of Bacteriology  1973;113(2):704-710.
There are two loci for resistance to the antibiotic kasugamycin (Ksg) in Escherichia coli. Mutations at ksgA resulted in 30S ribosomal subunit resistance to Ksg. The map location of ksgA was near minute 0.5: ksgA was 95% cotransducible with pdxA, and the apparent gene order was thr... ksgA... pdxA. Studies in stable ksgA/ksgA+ merodiploids showed that sensitivity was dominant over resistance. Mutations at a second gene (ksgB), located between minutes 25 and 39, resulted in phenotypic KsgR indistinguishable from ksgA mutations, but ribosomes from ksgB strains were sensitive to the drug in vitro. Spontaneous and induced mutations to KsgR were usually of the ksgA (ribosomal) type.
PMCID: PMC285284  PMID: 4570603
3.  The chlamydial functional homolog of KsgA confers kasugamycin sensitivity to Chlamydia trachomatis and impacts bacterial fitness 
BMC Microbiology  2009;9:279.
rRNA adenine dimethyltransferases, represented by the Escherichia coli KsgA protein, are highly conserved phylogenetically and are generally not essential for growth. They are responsible for the post-transcriptional transfer of two methyl groups to two universally conserved adenosines located near the 3'end of the small subunit rRNA and participate in ribosome maturation. All sequenced genomes of Chlamydia reveal a ksgA homolog in each species, including C. trachomatis. Yet absence of a S-adenosyl-methionine synthetase in Chlamydia, the conserved enzyme involved in the synthesis of the methyl donor S-adenosyl-L-methionine, raises a doubt concerning the activity of the KsgA homolog in these organisms.
Lack of the dimethylated adenosines following ksgA inactivation confers resistance to kasugamycin (KSM) in E. coli. Expression of the C. trachomatis L2 KsgA ortholog restored KSM sensitivity to the E. coli ksgA mutant, suggesting that the chlamydial KsgA homolog has specific rRNA dimethylase activity. C. trachomatis growth was sensitive to KSM and we were able to isolate a KSM resistant mutant of C. trachomatis containing a frameshift mutation in ksgA, which led to the formation of a shorter protein with no activity. Growth of the C. trachomatis ksgA mutant was negatively affected in cell culture highlighting the importance of the methylase in the development of these obligate intracellular and as yet genetically intractable pathogens.
The presence of a functional rRNA dimethylase enzyme belonging to the KsgA family in Chlamydia presents an excellent chemotherapeutic target with real potential. It also confirms the existence of S-adenosyl-methionine - dependent methylation reactions in Chlamydia raising the question of how these organisms acquire this cofactor.
PMCID: PMC2807437  PMID: 20043826
4.  Dimethyl Adenosine Transferase (KsgA) Deficiency in Salmonella enterica Serovar Enteritidis Confers Susceptibility to High Osmolarity and Virulence Attenuation in Chickens 
Applied and Environmental Microbiology  2013;79(24):7857-7866.
Dimethyl adenosine transferase (KsgA) performs diverse roles in bacteria, including ribosomal maturation and DNA mismatch repair, and synthesis of KsgA is responsive to antibiotics and cold temperature. We previously showed that a ksgA mutation in Salmonella enterica serovar Enteritidis results in impaired invasiveness in human and avian epithelial cells. In this study, we tested the virulence of a ksgA mutant (the ksgA::Tn5 mutant) of S. Enteritidis in orally challenged 1-day-old chickens. The ksgA::Tn5 mutant showed significantly reduced intestinal colonization and organ invasiveness in chickens compared to those of the wild-type (WT) parent. Phenotype microarray (PM) was employed to compare the ksgA::Tn5 mutant and its isogenic wild-type strain for 920 phenotypes at 28°C, 37°C, and 42°C. At chicken body temperature (42°C), the ksgA::Tn5 mutant showed significantly reduced respiratory activity with respect to a number of carbon, nitrogen, phosphate, sulfur, and peptide nitrogen nutrients. The greatest differences were observed in the osmolyte panel at concentrations of ≥6% NaCl at 37°C and 42°C. In contrast, no major differences were observed at 28°C. In independent growth assays, the ksgA::Tn5 mutant displayed a severe growth defect in high-osmolarity (6.5% NaCl) conditions in nutrient-rich (LB) and nutrient-limiting (M9 minimum salts) media at 42°C. Moreover, the ksgA::Tn5 mutant showed significantly reduced tolerance to oxidative stress, but its survival within macrophages was not impaired. Unlike Escherichia coli, the ksgA::Tn5 mutant did not display a cold-sensitivity phenotype; however, it showed resistance to kasugamycin and increased susceptibility to chloramphenicol. To the best of our knowledge, this is the first report showing the role of ksgA in S. Enteritidis virulence in chickens, tolerance to high osmolarity, and altered susceptibility to kasugamycin and chloramphenicol.
PMCID: PMC3837837  PMID: 24123731
5.  Transductional mapping of ksgB and a new Tn5-induced kasugamycin resistance gene, ksgD, in Escherichia coli K-12. 
Journal of Bacteriology  1981;145(2):914-919.
We have mapped the Escherichia coli ksgB gene to min 36.5, 0.8 min from man and 0.7 min from aroD. A new kasugamycin resistance (Ksgr) gene, ksgD, has been isolated, using a transposon, Tn5. ksgD::TN5 is 44% cotransducible with sbcA, unlinked to trp, and unlinked to man (by P1 transduction). The ksgD::Tn5 has a late time of entry from HfrB7 (PO43). These data place ksgD clockwise from sbcA (which enters early from HfrB7) at min 30.4. The reistance of ksgB ksgD single and double mutant strains has been quantitated. Single mutations, ksgB or ksgD, gave resistance to 600 micrograms of kasugamycin per ml, whereas a ksgB ksgD strain was able to grow in the presence of kasugamycin levels in excess of 3,000 micrograms/ml. This indicates that the mechanisms of resistance coded for by the two genes are independent and synergistic.
PMCID: PMC217198  PMID: 6257656
6.  Positive Growth Rate-Dependent Regulation of the pdxA, ksgA, and pdxB Genes of Escherichia coli K-12 
Journal of Bacteriology  2002;184(5):1359-1369.
We found that transcription of the pdxA and pdxB genes, which mediate steps in the biosynthesis of the essential coenzyme pyridoxal 5"-phosphate, and the ksgA gene, which encodes an rRNA modification enzyme and is partly cotranscribed with pdxA, is subject to positive growth rate regulation in Escherichia coli K-12. The amounts of the pdxA-ksgA cotranscript and pdxB- and ksgA-specific transcripts and expression from pdxA- and pdxB-lacZ fusions increased as the growth rate increased. The half-lives of ksgA- and pdxB-specific transcripts were not affected by the growth rate, whereas the half-life of the pdxA-ksgA cotranscript was too short to be measured accurately. A method of normalization was applied to determine the amount of mRNA synthesized per gene and the rate of protein accumulation per gene. Normalization removed an apparent anomaly at fast growth rates and demonstrated that positive regulation of pdxB occurs at the level of transcription initiation over the whole range of growth rates tested. RNA polymerase limitation and autoregulation could not account for the positive growth rate regulation of pdxA, pdxB, and ksgA transcription. On the other hand, growth rate regulation of the amount of the pdxA-ksgA cotranscript was abolished by a fis mutation, suggesting a role for the Fis protein. In contrast, the fis mutation had no effect on pdxB- or ksgA-specific transcript amounts. The amounts of the pdxA-ksgA cotranscript and ksgA-specific transcript were repressed in the presence of high intracellular concentrations of guanosine tetraphosphate; however, this effect was independent of relA function for the pdxA-ksgA cotranscript. Amounts of the pdxB-specific transcript remained unchanged during amino acid starvation in wild-type and relA mutant strains.
PMCID: PMC134838  PMID: 11844765
7.  Dissection of 16S rRNA Methyltransferase (KsgA) Function in Escherichia coli▿  
Journal of Bacteriology  2007;189(23):8510-8518.
A 16S rRNA methyltransferase, KsgA, identified originally in Escherichia coli is highly conserved in all living cells, from bacteria to humans. KsgA orthologs in eukaryotes possess functions in addition to their rRNA methyltransferase activity. E. coli Era is an essential GTP-binding protein. We recently observed that KsgA functions as a multicopy suppressor for the cold-sensitive cell growth of an era mutant [Era(E200K)] strain (Q. Lu and M. Inouye, J. Bacteriol. 180:5243-5246, 1998). Here we observed that although KsgA(E43A), KsgA(G47A), and KsgA(E66A) mutations located in the S-adenosylmethionine-binding motifs severely reduced its methyltransferase activity, these mutations retained the ability to suppress the growth defect of the Era(E200K) strain at a low temperature. On the other hand, a KsgA(R248A) mutation at the C-terminal domain that does not affect the methyltransferase activity failed to suppress the growth defect. Surprisingly, E. coli cells overexpressing wild-type KsgA, but not KsgA(R248A), were found to be highly sensitive to acetate even at neutral pH. Such growth inhibition also was observed in the presence of other weak organic acids, such as propionate and benzoate. These chemicals are known to be highly toxic at acidic pH by lowering the intracellular pH. We found that KsgA-induced cells had increased sensitivity to extreme acid conditions (pH 3.0) compared to that of noninduced cells. These results suggest that E. coli KsgA, in addition to its methyltransferase activity, has another unidentified function that plays a role in the suppression of the cold-sensitive phenotype of the Era(E200K) strain and that the additional function may be involved in the acid shock response. We discuss a possible mechanism of the KsgA-induced acid-sensitive phenotype.
PMCID: PMC2168933  PMID: 17890303
8.  Staphylococcus aureus and Escherichia coli have disparate dependences on KsgA for growth and ribosome biogenesis 
BMC Microbiology  2012;12:244.
The KsgA methyltransferase has been conserved throughout evolution, methylating two adenosines in the small subunit rRNA in all three domains of life as well as in eukaryotic organelles that contain ribosomes. Understanding of KsgA’s important role in ribosome biogenesis has been recently expanded in Escherichia coli; these studies help explain why KsgA is so highly conserved and also suggest KsgA’s potential as an antimicrobial drug target.
We have analyzed KsgA’s contribution to ribosome biogenesis and cell growth in Staphylococcus aureus. We found that deletion of ksgA in S. aureus led to a cold-sensitive growth phenotype, although KsgA was not as critical for ribosome biogenesis as it was shown to be in E. coli. Additionally, the ksgA knockout strain showed an increased sensitivity to aminoglycoside antibiotics. Overexpression of a catalytically inactive KsgA mutant was deleterious in the knockout strain but not the wild-type strain; this negative phenotype disappeared at low temperature.
This work extends the study of KsgA, allowing comparison of this aspect of ribosome biogenesis between a Gram-negative and a Gram-positive organism. Our results in S. aureus are in contrast to results previously described in E. coli, where the catalytically inactive protein showed a negative phenotype in the presence or absence of endogenous KsgA.
PMCID: PMC3534330  PMID: 23095113
KsgA; Ribosome biogenesis; Staphylococcus aureus; Escherichia coli; Methyltransferase
9.  KsgA, a 16S rRNA adenine methyltransferase, has a novel DNA glycosylase/AP lyase activity to prevent mutations in Escherichia coli 
Nucleic Acids Research  2009;37(7):2116-2125.
The 5-formyluracil (5-foU), a major mutagenic oxidative damage of thymine, is removed from DNA by Nth, Nei and MutM in Escherichia coli. However, DNA polymerases can also replicate past the 5-foU by incorporating C and G opposite the lesion, although the mechanism of correction of the incorporated bases is still unknown. In this study, using a borohydride-trapping assay, we identified a protein trapped by a 5-foU/C-containing oligonucleotide in an extract from E. coli mutM nth nei mutant. The protein was subsequently purified from the E. coli mutM nth nei mutant and was identified as KsgA, a 16S rRNA adenine methyltransferase. Recombinant KsgA also formed the trapped complex with 5-foU/C- and thymine glycol (Tg)/C-containing oligonucleotides. Furthermore, KsgA excised C opposite 5-foU, Tg and 5-hydroxymethyluracil (5-hmU) from duplex oligonucleotides via a β-elimination reaction, whereas it could not remove the damaged base. In contrast, KsgA did not remove C opposite normal bases, 7,8-dihydro-8-oxoguanine and 2-hydroxyadenine. Finally, the introduction of the ksgA mutation increased spontaneous mutations in E. coli mutM mutY and nth nei mutants. These results demonstrate that KsgA has a novel DNA glycosylase/AP lyase activity for C mispaired with oxidized T that prevents the formation of mutations, which is in addition to its known rRNA adenine methyltransferase activity essential for ribosome biogenesis.
PMCID: PMC2673420  PMID: 19223326
10.  The Gene for 16S rRNA Methyltransferase (ksgA) Functions as a Multicopy Suppressor for a Cold-Sensitive Mutant of Era, an Essential RAS-Like GTP-Binding Protein in Escherichia coli 
Journal of Bacteriology  1998;180(19):5243-5246.
Era, a Ras-like GTP-binding protein in Escherichia coli, has been shown to be essential for growth. However, its cellular functions still remain elusive. In this study, a genetic screening of an E. coli genomic library was performed to identify those genes which can restore the growth ability of a cold-sensitive mutant, Era(Cs) (E200K), at a restrictive temperature when expressed in a multicopy plasmid. Among eight suppressors isolated, six were located at 1 min of the E. coli genomic map, and the gene responsible for the suppression of Era(Cs) (E200K) was identified as the ksgA gene for 16S rRNA transmethylase, whose mutation causes a phenotype of resistance to kasugamycin, a translation initiation inhibitor. This is the first demonstration of suppression of impaired function of Era by overproduction of a functional enzyme. A possible mechanism of the suppression of the Era cold-sensitive phenotype by KsgA overproduction is discussed.
PMCID: PMC107565  PMID: 9748462
11.  Response of Methicillin-Resistant Staphylococcus aureus to Amicoumacin A 
PLoS ONE  2012;7(3):e34037.
Amicoumacin A exhibits strong antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), hence we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in transcription of genes specifying several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiholin-like product, which is induced in cells undergoing a collapse of Δψ. Consistent with the notion that LrgA modulates murein hydrolase activity, COL grown in the presence of amicoumacin A showed reduced autolysis, which was primarily caused by lower hydrolase activity. To gain further insight into the mechanism of action of amicoumacin A, a whole genome comparison of wild-type COL and amicoumacin A-resistant mutants isolated by a serial passage method was carried out. Single point mutations generating codon substitutions were uncovered in ksgA (encoding RNA dimethyltransferase), fusA (elongation factor G), dnaG (primase), lacD (tagatose 1,6-bisphosphate aldolase), and SACOL0611 (a putative glycosyl transferase). The codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance reside in separate domains and do not bring about cross resistance. Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation. Decreased rates of cellular metabolism including protein synthesis and DNA replication in resistant strains might allow cells to compensate for membrane dysfunction and thus increase cell survivability.
PMCID: PMC3316591  PMID: 22479511
12.  Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA 
Molecular microbiology  2008;70(5):1062-1075.
While the general blueprint of ribosome biogenesis is evolutionarily conserved, most details have diverged considerably. A striking exception to this divergence is the universally conserved KsgA/Dim1p enzyme family, which modifies two adjacent adenosines in the terminal helix of small subunit ribosomal RNA (rRNA). While localization of KsgA on 30S subunits (SSUs) and genetic interaction data have suggested that KsgA acts as a ribosome biogenesis factor, mechanistic details and a rationale for its extreme conservation are still lacking. To begin to address these questions we have characterized the function of E. coli KsgA in vivo using both a ksgA deletion strain and a methyltransferase deficient form of this protein. Our data reveals cold sensitivity and altered ribosomal profiles are associated with a ΔksgA genotype in E. coli. Our work also indicates that loss of KsgA alters 16S rRNA processing. These findings allow KsgAs role in SSU biogenesis to be integrated into the network of other identified factors. Moreover, a methyltransferase-inactive form of KsgA, which we show to be deleterious to cell growth, profoundly impairs ribosome biogenesis prompting discussion of KsgA as a possible anti-microbial drug target. These unexpected data suggest that methylation is a second layer of function for KsgA and that its critical role is as a supervisor of biogenesis of SSUs in vivo. These new findings and this proposed regulatory role offer a mechanistic explanation for the extreme conservation of the KsgA/Dim1p enzyme family.
PMCID: PMC2709978  PMID: 18990185
KsgA; Dim1p; rRNA processing; ribosome biogenesis
13.  Structural rearrangements in the active site of the Thermus thermophilus 16S rRNA methyltransferase KsgA in a binary complex with 5’-methylthio-adenosine 
Journal of molecular biology  2009;388(2):271-282.
Post-transcriptional modification of ribosomal RNA occurs in all kingdoms of life. The S-adenosyl-L-me-thionine-dependent methyltransferase KsgA introduces the most highly conserved ribosomal RNA modification, the dimethylation of A1518 and A1519 of 16S rRNA. Loss of this dimethylation confers resistance to the antibiotic kasugamycin. Here, we report biochemical studies and high-resolution crystal structures of KsgA from Thermus thermophilus. Methylation of 30S ribosomal subunits by T. thermophilus KsgA is more efficient at low concentrations of magnesium ions suggesting that partially unfolded RNA is the preferred substrate. The overall structure is similar to other methyltransferases but contains an additional α-helix in a novel N-terminal extension. Comparison of the apo-enzyme with complex structures with 5’-methylthioadenosine or adenosine bound in the cofactor-binding site reveal novel features when compared to related enzymes. Several mobile loop regions are observed that restrict access to the cofactor-binding site. In addition, the orientation of residues in the substrate-binding site indicates that conformational changes are required for binding two adjacent residues of the substrate rRNA.
PMCID: PMC2679894  PMID: 19285505
ribosome modification; 16S rRNA; 30S ribosomal subunit; rRNA methyltransferase; kasugamycin
14.  Genetic analysis of antibiotic resistance in Streptococcus pyogenes. 
Journal of Bacteriology  1978;133(2):852-859.
The genetics of antibiotic resistance in mutant strains of Streptococcus pyrogenes was studied. Utilizing a type 6 strain (9440) primarily resistant to strepttomycin (Strr), classes of mutant strains were isolated that were resistant to one of the following antibiotics: rifampin (Rifr), erythromycin (Eryr), thiostrepton (Tstr), spiramycin (Sprr), fusidic acid (Fusr), gramicidin (Grcr), ethidium bromide (Ebrr), kanamycin (Kanr), neomycin (Neor), oleandomycin (Oler), gentamicin (Genr), and novobiocin (Novr). Transduction experiments separated antibiotic resistance markers into two distinct groups: transducible markers, including Fusr, Bacr, Ksg+, Spcr, Eryr, Sprr, Rifr, Stlr, and Tstr (Bacr, Ksgr, Spcr, and Stlr refer to resistance to bacitracin, kasugamycin, spectinomycin, and streptolydigan, respectively), and nontransducible markers, including Grcr, Ebrr, Kanr, Neor, Oler, Genr, and Novr. By means of two- and three-point crosses, transducible markers (excluding tst) were located in three separate linkage groups. spr was found to be linked with ery and spc in the order spc-ery-spr, whereas in a separate linkage group the order was determined to be str-fus-bac-ksg. The third linkage group contained the rif and stl markers.
PMCID: PMC222097  PMID: 342510
15.  Autogenous regulation of the Escherichia coli ksgA gene at the level of translation. 
Journal of Bacteriology  1989;171(7):4002-4008.
Various plasmids that contain the Escherichia coli ksgA gene, which encodes a 16S rRNA adenosine dimethyltransferase (methylase), were constructed. In one of these plasmids, the DNA encoding the N-terminal part of the methylase was fused to the lacZ gene, and in another construct, the ksgA gene contained a deletion which resulted in a truncated version of the methylase. When a cell contained one plasmid directing the synthesis of the intact, active methylase and another plasmid encoding the methylase-beta-galactosidase protein, production of the latter product became strongly reduced. Likewise, synthesis of the truncated version of the methylase was diminished when the cell at the same time contained a plasmid producing the complete enzyme. These results were partly substantiated by in vitro experiments with a coupled transcription-translation assay system. By using a recently developed gel electrophoresis system for measuring protein-nucleic acid interactions, a specific binding of the ksgA methylase with its own mRNA could be established. Our results demonstrate that the expression of the ksgA gene can be, at least partly, autogenously controlled at the level of translation.
PMCID: PMC210154  PMID: 2500421
16.  Overlap between pdxA and ksgA in the complex pdxA-ksgA-apaG-apaH operon of Escherichia coli K-12. 
Journal of Bacteriology  1989;171(9):4767-4777.
We report that pdxA, which is required for de novo biosynthesis of pyridoxine (vitamin B6) and pyridoxal phosphate, belongs to an unusual, multifunctional operon. The pdxA gene was cloned in the same 3.5-kilobase BamHI-EcoRI restriction fragment that contains ksgA, which encodes the 16S rRNA modification enzyme m6(2)A methyltransferase, and apaH, which encodes diadenosine tetraphosphatase (ApppA hydrolase). Previously, Blanchin-Roland et al. showed that ksgA and apaH form a complex operon (Mol. Gen. Genet. 205:515-522, 1986). The pdxA gene was located on recombinant plasmids by subcloning, complementation, and insertion mutagenesis, and chromosomal insertions at five positions upstream from ksgA inactivated pdxA function. DNA sequence analysis and minicell translation experiments demonstrated that pdxA encoded a 35.1-kilodalton polypeptide and that the stop codon of pdxA overlapped the start codon of ksgA by 2 nucleotides. The translational start codon of pdxA was tentatively assigned based on polypeptide size and on the presence of a unique sequence that was also found near the translational start of PdxB. This conserved sequence may play a role in translational control of certain pyridoxine biosynthetic genes. RNase T2 mapping of chromosomal transcripts confirmed that pdxA and ksgA were members of the same complex operon, yet about half of ksgA transcripts arose in vivo under some culture conditions from an internal promoter mapped near the end of pdxA. Transcript analysis further suggested that pdxA is not the first gene in the operon. These structural features support the idea that pyridoxine-biosynthetic genes are members of complex operons, perhaps to interweave coenzyme biosynthesis genetically with other metabolic processes. The results are also considered in terms of ksgA expression.
PMCID: PMC210278  PMID: 2670894
17.  Identification of Attenuated Yersinia pseudotuberculosis Strains and Characterization of an Orogastric Infection in BALB/c Mice on Day 5 Postinfection by Signature-Tagged Mutagenesis 
Infection and Immunity  2001;69(5):2779-2787.
Yersinia pseudotuberculosis localizes to the distal ileum, cecum, and proximal colon of the gastrointestinal tract after oral infection. Using signature-tagged mutagenesis, we isolated 13 Y. pseudotuberculosis mutants that failed to survive in the cecum of mice after orogastric inoculation. Twelve of these mutants were also attenuated for replication in the spleen after intraperitoneal infection, whereas one strain, mutated the gene encoding invasin, replicated as well as wild-type bacteria in the spleen. Several mutations were in operons encoding components of the type III secretion system, including components involved in translocating Yop proteins into host cells. This indicates that one or more Yops may be necessary for survival in the gastrointestinal tract. Three mutants were defective in O-antigen biosynthesis; these mutants were also unable to invade epithelial cells as efficiently as wild-type Y. pseudotuberculosis. Several other mutations were in genes that had not previously been associated with growth in a host, including cls, ksgA, and sufl. In addition, using Y. pseudotuberculosis strains marked with signature tags, we counted the number of different bacterial clones that were present in the cecum, mesenteric lymph nodes, and spleen 5 days postinfection. We find barriers in the host animal that limit the number of bacteria that succeed in reaching and/or replicating in the mesenteric lymph nodes and spleen after breaching the gut mucosa.
PMCID: PMC98225  PMID: 11292689
18.  Diversity of Substitutions within or Adjacent to Conserved Amino Acid Motifs of Penicillin-Binding Protein 2X in Cephalosporin-Resistant Streptococcus pneumoniae Isolates 
The sequence of an approximately 1.1-kb DNA fragment of the pbp2x gene, which encodes the transpeptidase domain, was determined for 35 clinical isolates of Streptococcus pneumoniae for which the cefotaxime (CTX) MICs varied. Strains with substitutions within a conserved amino acid motif changing STMK to SAFK and a Leu-to-Val change just before the KSG motif were highly resistant to CTX (MIC, ≧2 μg/ml). Strains with substitutions adjacent to SSN or KSG motifs had low-level resistance. The amino acid substitutions were plotted on the three-dimensional crystallographic structure of the transpeptidase domain of PBP2X. Transformants containing pbp2x from strains with high-level CTX resistance increased the CTX MIC from 0.016 μg/ml to 0.5 to 1.0 μg/ml.
PMCID: PMC89251  PMID: 10223944
19.  In vitro selection of one-step mutants of Streptococcus pneumoniae resistant to different oral beta-lactam antibiotics is associated with alterations of PBP2x. 
Many oral penicillins and cephalosporins are used to treat clinical infections caused by Streptococcus pneumoniae. Therefore, using different beta-lactams as selectors, we estimated the frequencies of one-step mutations leading to resistance. Resistant mutants were obtained from penicillin-susceptible, intermediately resistant, and penicillin resistant strains. For cefixime, cefuroxime, cefpodoxime, cefotaxime, and ceftriaxone, the frequencies of mutation ranged from 10(-6) to 10(-8) when resistant mutants were selected at 2- to 8-fold the MIC, and the MICs increased 2- to 16-fold. For ampicillin, ampicillin-sulbactam, amoxicillin, amoxicillin-clavulanic acid, cefaclor, and loracarbef, the frequencies of mutation were about 10(-7) to 10(-8), and the MICs increased twofold at most. One to three resistance profiles of the resulting mutants were selected for each of the selecting antibiotics. Among those, some showed resistance to the cephalosporins associated with a 2- to 32-fold increase in susceptibility to the penicillins. Competition experiments showed a decreased affinity of PBP2x for cefpodoxime in all mutants. In some mutants that were more susceptible to amoxicillin, a decreased affinity of PBP2x for cefpodoxime was associated with an increased affinity for amoxicillin and a particular substitution of alanine for threonine at position 550 just after the KSG triad. From these results we infer (i) that among the beta-lactams tested the penicillins, cefaclor, and loracarbef selected one-step resistant mutants less frequently and that they achieved a lower level of resistance, and (ii) that mutants with different profiles may have acquired different point mutations in PBP2x.
PMCID: PMC163074  PMID: 8787897
20.  Structural analysis of kasugamycin inhibition of translation 
The prokaryotic ribosome is an important target of antibiotic action. We determined the X-ray structure of the aminoglycoside kasugamycin (Ksg) in complex with the Escherichia coli 70S ribosome at 3.5-Å resolution. The structure reveals that the drug binds within the messenger RNA channel of the 30S subunit between the universally conserved G926 and A794 nucleotides in 16S ribosomal RNA, which are sites of Ksg resistance. To our surprise, Ksg resistance mutations do not inhibit binding of the drug to the ribosome. The present structural and biochemical results indicate that inhibition by Ksg and Ksg resistance are closely linked to the structure of the mRNA at the junction of the peptidyl-tRNA and exit-tRNA sites (P and E sites).
PMCID: PMC2636691  PMID: 16998486
21.  Complete Sequences of Six Penicillin-Binding Protein Genes from 40 Streptococcus pneumoniae Clinical Isolates Collected in Japan 
All six penicillin-binding protein (PBP) genes, namely, pbp1a, pbp1b, pbp2a, pbp2b, pbp2x, and pbp3, of 40 Streptococcus pneumoniae clinical isolates, including penicillin-resistant S. pneumoniae isolates collected in Japan, were completely sequenced. The MICs of penicillin for these strains varied between 0.015 and 8 μg/ml. In PBP 2X, the Thr550Ala mutation close to the KSG motif was observed in only 1 of 40 strains, whereas the Met339Phe mutation in the STMK motif was observed in six strains. These six strains were highly resistant (MICs ≧ 2 μg/ml) to cefotaxime. The MICs of cefotaxime for 27 strains bearing the Thr338Ala mutation tended to increase, but the His394Leu mutation next to the SSN motif did not exist in these strains. In PBP 2B, the Thr451Ala/Phe/Ser and Glu481Gly mutations close to the SSN motif were observed in 24 strains, which showed penicillin resistance and intermediate resistance, and the Thr624Gly mutation close to the KTG motif was observed in 2 strains for which the imipenem MIC (0.5 μg/ml) was the highest imipenem MIC detected. In PBP 1A, the Thr371Ser/Ala mutation in the STMK motif was observed in all 13 strains for which the penicillin MICs were ≧1 μg/ml. In PBP 2A, the Thr411Ala mutation in the STIK motif was observed in one strain for which with the cefotaxime MIC (8 μg/ml) was the highest cefotaxime MIC detected. On the other hand, in PBPs 1B and 3, no mutations associated with resistance were observed. The results obtained here support the concept that alterations in PBPs 2B, 2X, and 1A are mainly involved in S. pneumoniae resistance to β-lactam antibiotics. Our findings also suggest that the Thr411Ala mutation in PBP 2A may be associated with β-lactam resistance.
PMCID: PMC415593  PMID: 15155228
22.  Characterization of Genetic and Phenotypic Diversity of Invasive Nontypeable Haemophilus influenzae  
Infection and Immunity  2005;73(9):5853-5863.
The ability of unencapsulated (nontypeable) Haemophilus influenzae (NTHi) to cause systemic disease in healthy children has been recognized only in the past decade. To determine the extent of similarity among invasive nontypeable isolates, we compared strain R2866 with 16 additional NTHi isolates from blood and spinal fluid, 17 nasopharyngeal or throat isolates from healthy children, and 19 isolates from middle ear aspirates. The strains were evaluated for the presence of several genetic loci that affect bacterial surface structures and for biochemical reactions that are known to differ among H. influenzae strains. Eight strains, including four blood isolates, shared several properties with R2866: they were biotype V (indole and ornithine decarboxylase positive, urease negative), contained sequence from the adhesin gene hia, and lacked a genetic island flanked by the infA and ksgA genes. Multilocus sequence typing showed that most biotype V isolates belonged to the same phylogenetic cluster as strain R2866. When present, the infA-ksgA island contains lipopolysaccharide biosynthetic genes, either lic2B and lic2C or homologs of the losA and losB genes described for Haemophilus ducreyi. The island was found in most nasopharyngeal and otitis isolates but was absent from 40% of invasive isolates. Overall, the 33 hmw-negative isolates were much more likely than hmw-containing isolates to have tryptophanase, ornithine decarboxylase, or lysine decarboxylase activity or to contain the hif genes. We conclude (i) that invasive isolates are genetically and phenotypically diverse and (ii) that certain genetic loci of NTHi are frequently found in association among NTHi strains.
PMCID: PMC1231076  PMID: 16113304
23.  Partial methylation of two adjacent adenosines in ribosomes from Euglena gracilis chloroplasts suggests evolutionary loss of an intermediate stage in the methyl-transfer reaction. 
Nucleic Acids Research  1984;12(23):9205-9208.
Bacterial, cytoplasmic and organellar ribosomes from a wide phylogenetic spectrum of organisms have a characteristic m6(2)Am6(2)A structure near the 3' end of the RNA of the small ribosomal subunit (SSU). We have studied one of the few exceptions to this extremely conserved post-transcriptionally modified sequence, i.e. dimethylation of only one of the two A's in chloroplasts from Euglena gracilis. It was established that only the A closest to the 5' end is dimethylated, the other one being unmodified. The methylation reaction was studied in vitro using ribosomes from a kasugamycin resistant mutant (ksgA) of Escherichia coli and purified methyl-transferase. Using limited amounts of the methyl donor S-adenosylmethionine (SAM) a partial level of methylation (50% of control) was attained. It is shown that in this case the 3' proximal A is dimethylated while the other is not. This suggests that dimethylation takes place in two successive stages. Apparently in E. gracilis chloroplasts the first stage of methylation does not occur.
PMCID: PMC320449  PMID: 6440121
24.  Characteristics of Plasmids in Multi-Drug-Resistant Enterobacteriaceae Isolated during Prospective Surveillance of a Newly Opened Hospital in Iraq 
PLoS ONE  2012;7(7):e40360.
Gram-negative multidrug-resistant (MDR) bacteria are major causes of nosocomial infections, and antibiotic resistance in these organisms is often plasmid mediated. Data are scarce pertaining to molecular mechanisms of antibiotic resistance in resource constrained areas such as Iraq.
Methodology/Principal Findings
In this study, all MDR Enterobacteriaceae (n = 38) and randomly selected non-MDR counterparts (n = 41) isolated from patients, healthcare workers and environmental surfaces in a newly opened hospital in Iraq were investigated to characterize plasmids found in these isolates and determine their contribution to antibiotic resistance. Our results demonstrated that MDR E. coli and K. pneumoniae isolates harbored significantly more (≥3) plasmids compared to their non-MDR counterparts, which carried ≤2 plasmids (p<0.01). Various large plasmids (∼52 to 100 kb) from representative isolates were confirmed to contain multiple resistance genes by DNA microarray analysis. Aminoglycoside (acc, aadA, aph, strA/B, and ksgA), β-lactam (blaTEM1, blaAMPC, blaCTX-M-15, blaOXA-1, blaVIM-2 and blaSHV), sulfamethoxazole/trimethoprim (sul/dfr), tetracycline (tet) and chloramphenicol (cat) resistance genes were detected on these plasmids. Additionally, multiple plasmids carrying multiple antibiotic resistance genes were found in the same host strain. Genetic transfer-associated genes were identified on the plasmids from both MDR and non-MDR isolates. Seven plasmid replicon types (FII, FIA, FIB, B/O, K, I1 and N) were detected in the isolates, while globally disseminated IncA/C and IncHI1 plasmids were not detected in these isolates.
This is the first report of the characteristics of the plasmids found in Enterobacteriaceae isolated following the opening of a new hospital in Iraq. The information provided here furthers our understanding of the mechanisms of drug resistance in this specific region and their evolutionary relationship with other parts of world. The large plasmids, carrying resistance genes and transfer-associated genes, may be potential factors for regional dissemination of antibiotic resistance.
PMCID: PMC3394789  PMID: 22808141
25.  Heterogeneity in Tandem Octanucleotides within Haemophilus influenzae Lipopolysaccharide Biosynthetic Gene losA Affects Serum Resistance  
Infection and Immunity  2006;74(6):3408-3414.
Haemophilus influenzae is subject to phase variation mediated by changes in the length of simple sequence repeat regions within several genes, most of which encode either surface proteins or enzymes involved in the synthesis of lipopolysaccharides (LPS). The translational repeat regions that have been described thus far all consist of tandemly repeated tetranucleotides. We describe an octanucleotide repeat region within a putative LPS biosynthetic gene, losA. Approximately 20 percent of nontypeable H. influenzae strains contain copies of losA and losB in a genetic locus flanked by infA and ksgA. Of 30 strains containing losA at this site, 24 contained 2 tandem copies of the octanucleotide CGAGCATA, allowing full-length translation of losA (on), and 6 strains contained 3, 4, 6, or 10 tandem copies (losA off). For a serum-sensitive strain, R3063, with losA off (10 repeat units), selection for serum-resistant variants yielded a heterogeneous population in which colonies with increased serum resistance had losA on (2, 8, or 11 repeat units), and colonies with unchanged sensitivity to serum had 10 repeats. Inactivation of losA in strains R3063 and R2846 (strain 12) by insertion of the cat gene decreased the serum resistance of these strains compared to losA-on variants and altered the electrophoretic mobility of LPS. We conclude that expression of losA, a gene that contributes to LPS structure and affects serum resistance, is determined by octanucleotide repeat variation.
PMCID: PMC1479228  PMID: 16714571

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