Hyunwoo Lee

The PmrA-regulated pmrC gene mediates phosphoethanolamine modification of lipid A and polymyxin resistance in Salmonella enterica.

The PmrA/PmrB regulatory system of Salmonella enterica controls the modification of lipid A with aminoarabinose and phosphoethanolamine. The aminoarabinose modification is required for resistance to the antibiotic polymyxin B, as mutations of the PmrA-activated pbg operon or ugd gene result in strains that lack aminoarabinose in their lipid A molecules and are more susceptible to polymyxin B. Additional PmrA-regulated genes appear to participate in polymyxin B resistance, as pbgP and ugd mutants are not as sensitive to polymyxin B as a pmrA mutant. Moreover, the role that the phosphoethanolamine modification of lipid A plays in the resistance to polymyxin B has remained unknown. Here we address both of these questions by establishing that the PmrA-activated pmrC gene encodes an inner membrane protein that is required for the incorporation of phosphoethanolamine into lipid A and for polymyxin B resistance. The PmrC protein consists of an N-terminal region with five transmembrane domains followed by a large periplasmic region harboring the putative enzymatic domain. A pbgP pmrC double mutant resembled a pmrA mutant both in its lipid A profile and in its susceptibility to polymyxin B, indicating that the PmrA-dependent modification of lipid A with aminoarabinose and phosphoethanolamine is responsible for PmrA-regulated polymyxin B resistance.

Increased Persistence in Escherichia coli Caused by Controlled Expression of Toxins or Other Unrelated Proteins

Bacterial populations contain persisters, cells which survive exposure to bactericidal antibiotics and other lethal factors. Persisters do not have a genetic resistance mechanism, and their means to tolerate killing remain unknown. In exponentially growing populations of Escherichia coli the frequency of persister formation usually is 10(-7) to 10(-5). It has been shown that cells overexpressing either of the toxic proteins HipA and RelE, both members of the bacterial toxin-antitoxin (TA) modules, have the ability to form more persisters, suggesting a specific role for these toxins in the mechanism of persistence. However, here we show that cells expressing proteins that are unrelated to TA modules but which become toxic when ectopically expressed, chaperone DnaJ and protein PmrC of Salmonella enterica, also form 100- to 1,000-fold more persisters. Thus, persistence is linked not only to toxicity caused by expression of HipA or dedicated toxins but also to expression of other unrelated proteins.

Nucleotide Biosynthesis Is Critical for Growth of Bacteria in Human Blood

Proliferation of bacterial pathogens in blood represents one of the most dangerous stages of infection. Growth in blood serum depends on the ability of a pathogen to adjust metabolism to match the availability of nutrients. Although certain nutrients are scarce in blood and need to be de novo synthesized by proliferating bacteria, it is unclear which metabolic pathways are critical for bacterial growth in blood. In this study, we identified metabolic functions that are essential specifically for bacterial growth in the bloodstream. We used two principally different but complementing techniques to comprehensively identify genes that are required for the growth of Escherichia coli in human serum. A microarray-based and a dye-based mutant screening approach were independently used to screen a library of 3,985 single-gene deletion mutants in all non-essential genes of E. coli (Keio collection). A majority of the mutants identified consistently by both approaches carried a deletion of a gene involved in either the purine or pyrimidine nucleotide biosynthetic pathway and showed a 20- to 1,000-fold drop in viable cell counts as compared to wild-type E. coli after 24 h of growth in human serum. This suggests that the scarcity of nucleotide precursors, but not other nutrients, is the key limitation for bacterial growth in serum. Inactivation of nucleotide biosynthesis genes in another Gram-negative pathogen, Salmonella enterica, and in the Gram-positive pathogen Bacillus anthracis, prevented their growth in human serum. The growth of the mutants could be rescued by genetic complementation or by addition of appropriate nucleotide bases to human serum. Furthermore, the virulence of the B. anthracis purE mutant, defective in purine biosynthesis, was dramatically attenuated in a murine model of bacteremia. Our data indicate that de novo nucleotide biosynthesis represents the single most critical metabolic function for bacterial growth in blood and reveal the corresponding enzymes as putative antibiotic targets for the treatment of bloodstream infections.

Molecular sieve mechanism of selective release of cytoplasmic proteins by osmotically shocked Escherichia coli.

Escherichia coli cells, the outer membrane of which is permeabilized with EDTA, release a specific subset of cytoplasmic proteins upon a sudden drop in osmolarity in the surrounding medium. This subset includes EF-Tu, thioredoxin, and DnaK among other proteins, and comprises approximately 10% of the total bacterial protein content. As we demonstrate here, the same proteins are released from electroporated E. coli cells pretreated with EDTA. Although known for several decades, the phenomenon of selective release of proteins has received no satisfactory explanation. Here we show that the subset of released proteins is almost identical to the subset of proteins that are able to pass through a 100-kDa-cutoff cellulose membrane upon molecular filtration of an E. coli homogenate. This finding indicates that in osmotically shocked or electroporated bacteria, proteins are strained through a molecular sieve formed by the transiently damaged bacterial envelope. As a result, proteins of small native sizes are selectively released, whereas large proteins and large protein complexes are retained by bacterial cells.

The Bacillus anthracis Protein MprF Is Required for Synthesis of Lysylphosphatidylglycerols and for Resistance to Cationic Antimicrobial Peptides

During inhalational anthrax, Bacillus anthracis survives and replicates in alveolar macrophages, followed by rapid invasion into the hosts bloodstream, where it multiplies to cause heavy bacteremia. B. anthracis must therefore defend itself from host immune functions encountered during both the intracellular and the extracellular stages of anthrax infection. In both of these niches, cationic antimicrobial peptides are an essential component of the hosts innate immune response that targets B. anthracis. However, the genetic determinants of B. anthracis contributing to resistance to these peptides are largely unknown. Here we generated Tn917 transposon mutants in the DeltaANR strain (pXO1- pXO2-) of B. anthracis and screened them for altered protamine susceptibility. A protamine-sensitive mutant identified carried the transposon inserted in the BA1486 gene encoding a putative membrane protein homologous to MprF known in several gram-positive pathogens. A mutant strain with the BAS1375 gene (the orthologue of BA1486) deleted in the Sterne 34F2 strain (pXO1+ pXO2-) of B. anthracis exhibited hypersusceptibility not only to protamine but also to alpha-helical cathelicidin LL-37 and beta-sheet defensin human neutrophil peptide 1 compared to the wild-type Sterne strain. Analysis of membrane lipids using isotopic labeling demonstrated that the BAS1375 deletion mutant is unable to synthesize lysinylated phosphatidylglycerols, and this defect is rescued by genetic complementation. Further, we determined the structures of these lysylphosphatidylglycerols by using various mass spectrometric analyses. These results demonstrate that in B. anthracis a functional MprF is required for the biosynthesis of lysylphosphatidylglycerols, which is critical for resistance to cationic antimicrobial peptides.

Identification of the P3 Promoter and Distinct Roles of the Two Promoters of the SaeRS Two-Component System in Staphylococcus aureus

In Staphylococcus aureus, the SaeRS two-component system (TCS) encoded by the saePQRS operon controls expression of major virulence factors, such as coagulase and alpha-hemolysin. The saePQRS operon has two promoters: P1 and P3. The P1 promoter, a strong promoter, is autoinduced and can transcribe all four genes. Compared with P1, P3 shows fairly low but constitutive promoter activity, and it transcribes only saeR and saeS, the two genes encoding response regulator SaeR and sensor kinase SaeS. However, the role of each promoter in sae signaling has not been rigorously defined. In this study, we found that the genuine transcription start site (TSS) of P3 is located 78 nucleotides downstream of the previously reported TSS. Subsequently, the P3 promoter sequence was identified and validated by mutagenesis analyses. Deletion of the saePQ region including the P1 promoter did not significantly alter the expression patterns of coagulase and alpha-hemolysin, two well-known sae target genes. Due to its L18P substitution in a transmembrane domain, SaeS in strain Newman has a constitutive kinase activity. Interestingly, the mutation also rendered the protein unstable, but the protein stability was restored by SaeQ, suggesting a possible SaeQ-SaeS interaction. Ironically, the same mutation seems to increase mRNA stability. SaeR appears to be stabilized by SaeS, possibly by a protein-protein interaction. Chromosomal mutation of P1 did not affect the expression pattern of coagulase and alpha-hemolysin. Based on these results, we conclude that transcription of saeRS from P3 is sufficient for target gene activation and that P1 is not involved in the activation.

Transcriptomic and phenotypic analysis of paralogous spx gene function in Bacillus anthracis Sterne

Spx of Bacillus subtilis is a redox‐sensitive protein, which, under disulfide stress, interacts with RNA polymerase to activate genes required for maintaining thiol homeostasis. Spx orthologs are highly conserved among low %GC Gram‐positive bacteria, and often exist in multiple paralogous forms. In this study, we used B. anthracis Sterne, which harbors two paralogous spx genes, spxA1 and spxA2, to examine the phenotypes of spx null mutations and to identify the genes regulated by each Spx paralog. Cells devoid of spxA1 were sensitive to diamide and hydrogen peroxide, while the spxA1 spoxA2 double mutant was hypersensitive to the thiol‐specific oxidant, diamide. Bacillus anthracis Sterne strains expressing spxA1DD or spxA2DD alleles encoding protease‐resistant products were used in microarray and quantitative real‐time polymerase chain reaction (RT‐qPCR) analyses in order to uncover genes under SpxA1, SpxA2, or SpxA1/SpxA2 control. Comparison of transcriptomes identified many genes that were upregulated when either SpxA1DD or SpxA2DD was produced, but several genes were uncovered whose transcript levels increased in only one of the two SpxADD‐expression strains, suggesting that each Spx paralog governs a unique regulon. Among genes that were upregulated were those encoding orthologs of proteins that are specifically involved in maintaining intracellular thiol homeostasis or alleviating oxidative stress. Some of these genes have important roles in B. anthracis pathogenesis, and a large number of upregulated hypothetical genes have no homology outside of the B. cereus/thuringiensis group. Microarray and RT‐qPCR analyses also unveiled a regulatory link that exists between the two spx paralogous genes. The data indicate that spxA1 and spxA2 are transcriptional regulators involved in relieving disulfide stress but also control a set of genes whose products function in other cellular processes.

Thermoregulation of Capsule Production by Streptococcus pyogenes

The capsule of Streptococcus pyogenes serves as an adhesin as well as an anti-phagocytic factor by binding to CD44 on keratinocytes of the pharyngeal mucosa and the skin, the main entry sites of the pathogen. We discovered that S. pyogenes HSC5 and MGAS315 strains are further thermoregulated for capsule production at a post-transcriptional level in addition to the transcriptional regulation by the CovRS two-component regulatory system. When the transcription of the hasABC capsular biosynthetic locus was de-repressed through mutation of the covRS system, the two strains, which have been used for pathogenesis studies in the laboratory, exhibited markedly increased capsule production at sub-body temperature. Employing transposon mutagenesis, we found that CvfA, a previously identified membrane-associated endoribonuclease, is required for the thermoregulation of capsule synthesis. The mutation of the cvfA gene conferred increased capsule production regardless of temperature. However, the amount of the capsule transcript was not changed by the mutation, indicating that a post-transcriptional regulator mediates between CvfA and thermoregulated capsule production. When we tested naturally occurring invasive mucoid strains, a high percentage (11/53, 21%) of the strains exhibited thermoregulated capsule production. As expected, the mucoid phenotype of these strains at sub-body temperature was due to mutations within the chromosomal covRS genes. Capsule thermoregulation that exhibits high capsule production at lower temperatures that occur on the skin or mucosal surface potentially confers better capability of adhesion and invasion when S. pyogenes penetrates the epithelial surface.

Identification of Unannotated Small Genes in Salmonella

Increasing evidence indicates that many, if not all, small genes encoding proteins ≤100 aa are missing in annotations of bacterial genomes currently available. To uncover unannotated small genes in the model bacterium Salmonella enterica Typhimurium 14028s, we used the genomic technique ribosome profiling, which provides a snapshot of all mRNAs being translated (translatome) in a given growth condition. For comprehensive identification of unannotated small genes, we obtained Salmonella translatomes from four different growth conditions: LB, MOPS rich defined medium, and two infection-relevant conditions low Mg2+ (10 µM) and low pH (5.8). To facilitate the identification of small genes, ribosome profiling data were analyzed in combination with in silico predicted putative open reading frames and transcriptome profiles. As a result, we uncovered 130 unannotated ORFs. Of them, 98% were small ORFs putatively encoding peptides/proteins ≤100 aa, and some of them were only expressed in the infection-relevant low Mg2+ and/or low pH condition. We validated the expression of 25 of these ORFs by western blot, including the smallest, which encodes a peptide of 7 aa residues. Our results suggest that many sequenced bacterial genomes are underannotated with regard to small genes and their gene annotations need to be revised.

Monitoring of gene knockouts: genome-wide profiling of conditionally essential genes

We have developed a new microarray-based genetic technique, named MGK (Monitoring of Gene Knockouts), for genome-wide identification of conditionally essential genes. MGK identified bacterial genes that are critical for fitness in the absence of aromatic amino acids, and was further applied to identify genes whose inactivation causes bacterial cell death upon exposure to the bacteriostatic antibiotic chloramphenicol. Our findings suggest that MGK can serve as a robust tool in functional genomics studies.