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1.  Ectopic Overexpression of Wild-Type and Mutant hipA Genes in Escherichia coli: Effects on Macromolecular Synthesis and Persister Formation 
Journal of Bacteriology  2006;188(11):3826-3836.
Persistence is an epigenetic trait that allows a small fraction of bacteria, approximately one in a million, to survive prolonged exposure to antibiotics. In Escherichia coli an increased frequency of persisters, called “high persistence,” is conferred by mutations in the hipA gene, which encodes the toxin entity of the toxin-antitoxin module hipBA. The high-persistence allele hipA7 was originally identified because of its ability to confer high persistence, but little is known about the physiological role of the wild-type hipA gene. We report here that the expression of wild-type hipA in excess of hipB inhibits protein, RNA, and DNA synthesis in vivo. However, unlike the RelE and MazF toxins, HipA had no effect on protein synthesis in an in vitro translation system. Moreover, the expression of wild-type hipA conferred a transient dormant state (persistence) to a sizable fraction of cells, whereas the rest of the cells remained in a prolonged dormant state that, under appropriate conditions, could be fully reversed by expression of the cognate antitoxin gene hipB. In contrast, expression of the mutant hipA7 gene in excess of hipB did not markedly inhibit protein synthesis as did wild-type hipA and yet still conferred persistence to ca. 10% of cells. We propose that wild-type HipA, upon release from HipB, is able to inhibit macromolecular synthesis and induces a bacteriostatic state that can be reversed by expression of the hipB gene. However, the ability of the wild-type hipA gene to generate a high frequency of persisters, equal to that conferred by the hipA7 allele, may be distinct from the ability to block macromolecular synthesis.
doi:10.1128/JB.01740-05
PMCID: PMC1482909  PMID: 16707675
2.  Molecular Mechanisms of HipA Mediated Multidrug Tolerance and its Neutralization by HipB 
Science (New York, N.Y.)  2009;323(5912):396-401.
Summary
Bacterial multidrug tolerance is largely responsible for the inability of antibiotics to eradicate infections and is caused by a small population of dormant bacteria called persisters. HipA is a critical Escherichia coli persistence factor that is normally neutralized by HipB, a transcription repressor, which also regulates hipBA expression. Here we report multiple structures of HipA and a HipA-HipB-DNA complex. HipA has a eukaryotic Ser/Thr kinase-like fold and can phosphorylate the translation factor, EF-Tu, suggesting a persistence mechanism via cell stasis. The HipA-HipB-DNA structure reveals the HipB-operator binding mechanism, ~70° DNA bending and unexpected HipA-DNA contacts. Dimeric HipB interacts with two HipA molecules to inhibit its kinase activity through sequestration and conformational inactivation. Combined, these studies suggest mechanisms for HipA-mediated persistence and its neutralization by HipB.
doi:10.1126/science.1163806
PMCID: PMC2764309  PMID: 19150849
3.  Regulation of the Escherichia coli HipBA Toxin-Antitoxin System by Proteolysis 
PLoS ONE  2012;7(6):e39185.
Bacterial populations produce antibiotic-tolerant persister cells. A number of recent studies point to the involvement of toxin/antitoxin (TA) modules in persister formation. hipBA is a type II TA module that codes for the HipB antitoxin and the HipA toxin. HipA is an EF-Tu kinase, which causes protein synthesis inhibition and dormancy upon phosphorylation of its substrate. Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases. We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon- background. These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis. Moreover, we demonstrated that degradation of HipB is dependent on the presence of an unstructured carboxy-terminal stretch of HipB that encompasses the last 16 amino acid residues. Further, substitution of the conserved carboxy-terminal tryptophan of HipB to alanine or even the complete removal of this 16 residue fragment did not alter the affinity of HipB for hipBA operator DNA or for HipA indicating that the major role of this region of HipB is to control HipB degradation and hence HipA-mediated persistence.
doi:10.1371/journal.pone.0039185
PMCID: PMC3376134  PMID: 22720069
4.  Autoregulation of hip, an operon that affects lethality due to inhibition of peptidoglycan or DNA synthesis. 
Journal of Bacteriology  1994;176(13):4081-4091.
The hip locus of Escherichia coli affects the frequency of persistence to the lethal consequences of selective inhibition of either DNA or peptidoglycan synthesis. Regulation of the hip operon, which consists of a regulatory region and two genes, hipB and hipA, was examined with strains containing a hip-lac transcriptional fusion placed in single copy at the lambda att site. Disruption of the hip locus increased activity from the fusion 16-fold. Repression was restored by supplying HipB in trans. HipB was overexpressed and purified. On the basis of gel filtration and cross-linking studies, HipB is a dimer in solution. Sequence analysis revealed that HipB is a Cro-like DNA-binding protein. The interaction of HipB with the hip regulatory region was examined by gel retardation, DNase I protection, and methylation protection studies. HipB binds with a Kapp (K apparent) of 40 pM to four operator sites with the conserved sequence TATCCN8GGATA (N represents any nucleotide). Binding to the operators is nearly simultaneous and appears to be cooperative. Analysis of the role of HipA in the regulation of the hip operon is complicated by the toxicity of HipA in the absence of HipB. Strains disrupted in hipB but not in hipA could not be recovered. Moreover, hipA-containing plasmids cannot be replicated in strains defective in or lacking hipB. HipA is found exclusively in a tight complex with HipB. Although disruption of hipA slightly increased expression from the hip-lac fusion, in vitro studies suggest that HipA does not bind to the hip regulatory region directly but indirectly via HipB.
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PMCID: PMC205607  PMID: 8021189
5.  HipA-Triggered Growth Arrest and β-Lactam Tolerance in Escherichia coli Are Mediated by RelA-Dependent ppGpp Synthesis 
Journal of Bacteriology  2013;195(14):3173-3182.
Persistence is a phenomenon whereby a subpopulation of bacterial cells enters a transient growth-arrested state that confers antibiotic tolerance. While entrance into persistence has been linked to the activities of toxin proteins, the molecular mechanisms by which toxins induce growth arrest and the persistent state remain unclear. Here, we show that overexpression of the protein kinase HipA in Escherichia coli triggers growth arrest by activating synthesis of the alarmone guanosine tetraphosphate (ppGpp) by the enzyme RelA, a signal typically associated with amino acid starvation. We further demonstrate that chemically suppressing ppGpp synthesis with chloramphenicol relieves inhibition of DNA replication initiation and RNA synthesis in HipA-arrested cells and restores vulnerability to β-lactam antibiotics. HipA-arrested cells maintain glucose uptake and oxygen consumption and accumulate amino acids as a consequence of translational inhibition. We harness the active metabolism of HipA-arrested cells to provide a bacteriophage-resistant platform for the production of biotechnologically relevant compounds, which may represent an innovative solution to the costly problem of phage contamination in industrial fermentations.
doi:10.1128/JB.02210-12
PMCID: PMC3697643  PMID: 23667235
6.  Molecular cloning and expression of hipA, a gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis. 
Journal of Bacteriology  1986;166(2):399-403.
The hipA gene at 33.8 min on the Escherichia coli chromosome controls the frequency of persistence upon inhibition of murein synthesis; for strains bearing hipA+ the frequency is 10(-6), and for hipA- strains the frequency is 10(-2). hip+ has been cloned by selection for a kanamycin resistance determinant at 33.9 min. hipA+ is dominant over hipA- in both recA+ and recA- backgrounds. The smallest DNA insert which contains hipA+, as determined by the ability of the plasmids to complement hipA- strains, is 1,885 base pairs. Both orientations of hipA+ are obtained when the cloning site of vector is remote from strong promoters; both orientations complement hipA-, and both encode a unique peptide of 50,000 Mr. The probable direction of transcription has been deduced from the pattern of peptides encoded by plasmids from which either end of the insert and adjacent vector sequences have been deleted. This information and the recovery of only one orientation of hipA+ when the cloning site is close to a strong promoter suggest that a high level of expression of the gene is not tolerated by E. coli.
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PMCID: PMC214618  PMID: 3516974
7.  Specialized Persister Cells and the Mechanism of Multidrug Tolerance in Escherichia coli 
Journal of Bacteriology  2004;186(24):8172-8180.
Bacterial populations produce persisters, cells that neither grow nor die in the presence of bactericidal agents, and thus exhibit multidrug tolerance (MDT). The mechanisms of MDT and the nature of persisters have remained elusive. Our previous research has shown that persisters are largely responsible for the recalcitrance of biofilm infections. A general method for isolating persisters was developed, based on lysis of regular cells by ampicillin. A gene expression profile of persisters contained toxin-antitoxin (TA) modules and other genes that can block important cellular functions such as translation. Bactericidal antibiotics kill cells by corrupting the target function (for example, aminoglycosides interrupt translation, producing toxic peptides). We reasoned that inhibition of translation will lead to a shutdown of cellular functions, preventing antibiotics from corrupting their targets, giving rise to MDT persister cells. Overproduction of the RelE toxin, an inhibitor of translation, caused a sharp increase in persisters. Functional expression of a putative HipA toxin also increased persisters, while deletion of the hipBA module caused a sharp decrease in persisters in both stationary and biofilm populations. HipA is thus the first validated persister-MDT gene. We suggest that random fluctuation in the levels of MDT proteins leads to the formation of rare persister cells. The function of these specialized dormant cells is to ensure the survival of the population in the presence of lethal factors.
doi:10.1128/JB.186.24.8172-8180.2004
PMCID: PMC532439  PMID: 15576765
8.  Structure and organization of hip, an operon that affects lethality due to inhibition of peptidoglycan or DNA synthesis. 
Journal of Bacteriology  1991;173(18):5732-5739.
High-frequency persistence to the lethal effects of inhibition of either DNA or peptidoglycan synthesis, the Hip phenotype, results from mutations at the hip locus of Escherichia coli K-12. The nucleotide sequence of DNA fragments which complement these mutations revealed an operon consisting of a possible regulatory region, including sequences with modest homology to an E. coli promoter, and two open reading frames which are translated both in vitro and in vivo. The stop codon of a 264-bp open reading frame, hipB, and the start codon of a 1,320-bp open reading frame, hipA, share an adenine residue. Assays of promoter strength, the location of the probable promoter with respect to the start of transcription, and codon usage all indicate that hipB and hipA are weakly expressed genes. The activity of the promoter is impaired by an adjacent downstream sequence which includes the coding region of hipB. The impairment is partially relieved by insertion of a premature translation termination signal within the coding region of hipB, suggesting involvement of the HipB protein in the regulation of this promoter. The arrangement of hipB and hipA within the operon and the toxicity of hipA for strains defective in or lacking hipB suggest an important interaction between the products of these genes.
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PMCID: PMC208304  PMID: 1715862
9.  Increased Persistence in Escherichia coli Caused by Controlled Expression of Toxins or Other Unrelated Proteins 
Journal of Bacteriology  2006;188(10):3494-3497.
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.
doi:10.1128/JB.188.10.3494-3497.2006
PMCID: PMC1482871  PMID: 16672603
10.  Transcriptional cross-activation between toxin-antitoxin systems of Escherichia coli 
BMC Microbiology  2013;13:45.
Background
Bacterial toxin-antitoxin (TA) systems are formed by potent regulatory or suicide factors (toxins) and their short-lived inhibitors (antitoxins). Antitoxins are DNA-binding proteins and auto-repress transcription of TA operons. Transcription of multiple TA operons is activated in temporarily non-growing persister cells that can resist killing by antibiotics. Consequently, the antitoxin levels of persisters must have been dropped and toxins are released of inhibition.
Results
Here, we describe transcriptional cross-activation between different TA systems of Escherichia coli. We find that the chromosomal relBEF operon is activated in response to production of the toxins MazF, MqsR, HicA, and HipA. Expression of the RelE toxin in turn induces transcription of several TA operons. We show that induction of mazEF during amino acid starvation depends on relBE and does not occur in a relBEF deletion mutant. Induction of TA operons has been previously shown to depend on Lon protease which is activated by polyphospate accumulation. We show that transcriptional cross-activation occurs also in strains deficient for Lon, ClpP, and HslV proteases and polyphosphate kinase. Furthermore, we find that toxins cleave the TA mRNA in vivo, which is followed by degradation of the antitoxin-encoding fragments and selective accumulation of the toxin-encoding regions. We show that these accumulating fragments can be translated to produce more toxin.
Conclusion
Transcriptional activation followed by cleavage of the mRNA and disproportionate production of the toxin constitutes a possible positive feedback loop, which can fire other TA systems and cause bistable growth heterogeneity. Cross-interacting TA systems have a potential to form a complex network of mutually activating regulators in bacteria.
doi:10.1186/1471-2180-13-45
PMCID: PMC3598666  PMID: 23432955
Toxin-antitoxin systems; Transcriptional regulation; Regulatory network; mRNA stability; Persisters
11.  The bacterial antitoxin HipB establishes a ternary complex with operator DNA and phosphorylated toxin HipA to regulate bacterial persistence 
Nucleic Acids Research  2014;42(15):10134-10147.
Nearly all bacteria exhibit a type of phenotypic growth described as persistence that is thought to underlie antibiotic tolerance and recalcitrant chronic infections. The chromosomally encoded high-persistence (Hip) toxin–antitoxin proteins HipASO and HipBSO from Shewanella oneidensis, a proteobacterium with unusual respiratory capacities, constitute a type II toxin–antitoxin protein module. Here we show that phosphorylated HipASO can engage in an unexpected ternary complex with HipBSO and double-stranded operator DNA that is distinct from the prototypical counterpart complex from Escherichia coli. The structure of HipBSO in complex with operator DNA reveals a flexible C-terminus that is sequestered by HipASO in the ternary complex, indicative of its role in binding HipASO to abolish its function in persistence. The structure of HipASO in complex with a non-hydrolyzable ATP analogue shows that HipASO autophosphorylation is coupled to an unusual conformational change of its phosphorylation loop. However, HipASO is unable to phosphorylate the translation factor Elongation factor Tu, contrary to previous reports, but in agreement with more recent findings. Our studies suggest that the phosphorylation state of HipA is an important factor in persistence and that the structural and mechanistic diversity of HipAB modules as regulatory factors in bacterial persistence is broader than previously thought.
doi:10.1093/nar/gku665
PMCID: PMC4150777  PMID: 25056321
12.  The Three RelE Homologs of Mycobacterium tuberculosis Have Individual, Drug-Specific Effects on Bacterial Antibiotic Tolerance▿ †  
Journal of Bacteriology  2010;192(5):1279-1291.
In Escherichia coli, expression of the RelE and HipA toxins in the absence of their cognate antitoxins has been associated with generating multidrug-tolerant “persisters.” Here we show that unlike persisters of E. coli, persisters of Mycobacterium tuberculosis selected with one drug do not acquire cross-resistance to other classes of drugs. M. tuberculosis has three homologs of RelE arranged in operons with their apparent antitoxins. Each toxin individually arrests growth of both M. tuberculosis and E. coli, an effect that is neutralized by coexpression of the cognate antitoxin. Overexpression or deletion of each of the RelE toxins had a toxin- and drug-specific effect on the proportion of bacilli surviving antibiotic killing. All three toxins were upregulated in vivo, but none of the deletions affected survival during murine infection. RelE2 overexpression increased bacterial survival rates in the presence of rifampin in vitro, while deletion significantly decreased survival rates. Strikingly, deletion of this toxin had no discernible effect on the level of persisters seen in rifampin-treated mice. Our results suggest that, in vivo, RelE-generated persisters are unlikely to play a significant role in the generation of bacilli that survive in the face of multidrug therapy or in the generation of multidrug-resistant M. tuberculosis.
doi:10.1128/JB.01285-09
PMCID: PMC2820853  PMID: 20061486
13.  Joint Tolerance to β-Lactam and Fluoroquinolone Antibiotics in Escherichia coli Results from Overexpression of hipA 
Antimicrobial Agents and Chemotherapy  1998;42(12):3282-3284.
The basis of joint tolerance to β-lactam and fluoroquinolone antibiotics in Escherichia coli mediated by hipA was examined. An antibiotic tolerance phenotype was produced by overexpression of hipA under conditions that did not affect the growth rate of the organism. Overexpressing hipA probably decreases the period in which bacteria are susceptible to the antibiotics by temporarily affecting some aspect of chromosome replication or cell division.
PMCID: PMC106036  PMID: 9835528
14.  hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis. 
Journal of Bacteriology  1983;155(2):768-775.
Except for a small fraction of persisters, 10(-6) to 10(-5), Escherichia coli K-12 is killed by prolonged inhibition of murein synthesis. The progeny of persisters are neither more resistant to inhibition of murein synthesis nor more likely to persist than normal cells. Mutants have been isolated in which a larger fraction, 10(-2), persists. The persistent response of the mutants, Hip (high persistence), is to inhibition of murein synthesis at early or late steps by antibiotics (phosphomycin, cycloserine, and ampicillin) or by metabolic block (starvation for diaminopimelic acid). Killing of the parent strain by each of the four inhibitors has two phases: The first is rapid and lasts about 30 min; the second is slower, but still substantial, and lasts 3 to 4 h. The first phase also occurs in the Hip mutants, but then viability of the mutants remains constant after about 30 min. Neither tolerance, resistance, impaired growth, nor reversion of spheroplasts accounts for high-frequency persistence. Two of the mutations map at 33.8 min in a region containing few other recognized functions. This position and the phenotypes define hipA as a newly recognized gene. Transposons Tn5 and Tn10 have been inserted close to hipA making it possible to explore the molecular genetics of persistence, a long recognized but poorly understood phenomenon.
PMCID: PMC217749  PMID: 6348026
15.  The Escherichia coli mqsR and ygiT Genes Encode a New Toxin-Antitoxin Pair▿ † 
Journal of Bacteriology  2010;192(11):2908-2919.
Toxin-antitoxin (TA) systems are plasmid- or chromosome-encoded protein complexes composed of a stable toxin and a short-lived inhibitor of the toxin. In cultures of Escherichia coli, transcription of toxin-antitoxin genes was induced in a nondividing subpopulation of bacteria that was tolerant to bactericidal antibiotics. Along with transcription of known toxin-antitoxin operons, transcription of mqsR and ygiT, two adjacent genes with multiple TA-like features, was induced in this cell population. Here we show that mqsR and ygiT encode a toxin-antitoxin system belonging to a completely new family which is represented in several groups of bacteria. The mqsR gene encodes a toxin, and ectopic expression of this gene inhibits growth and induces rapid shutdown of protein synthesis in vivo. ygiT encodes an antitoxin, which protects cells from the effects of MqsR. These two genes constitute a single operon which is transcriptionally repressed by the product of ygiT. We confirmed that transcription of this operon is induced in the ampicillin-tolerant fraction of a growing population of E. coli and in response to activation of the HipA toxin. Expression of the MqsR toxin does not kill bacteria but causes reversible growth inhibition and elongation of cells.
doi:10.1128/JB.01266-09
PMCID: PMC2876487  PMID: 20233923
16.  Conditional impairment of cell division and altered lethality in hipA mutants of Escherichia coli K-12. 
Journal of Bacteriology  1988;170(8):3321-3326.
Mutations in hipA, a gene of Escherichia coli K-12, greatly reduce the lethality of selective inhibition of peptidoglycan synthesis. These mutations have also been found to reduce the lethality that accompanies either selective inhibition of DNA synthesis or heat shock of strains defective in htpR. In addition, the mutant alleles of hipA are responsible for a reversible cold-sensitive block in cell division and synthesis of macromolecules, particularly peptidoglycan. Recombination between the chromosome of hipA mutants and plasmids containing noncomplementing fragments of hipA+ revealed that the mutations responsible for both cold sensitivity and reduced lethality were probably identical and, in any case, lay within the first 360 base pairs of the coding region of hipA, probably within the first 50 base pairs. We suggest that the pleiotropic effects of mutations in hipA reflect the involvement of this gene in cell division.
PMCID: PMC211297  PMID: 3042747
17.  Mutants of Escherichia coli K-12 exhibiting reduced killing by both quinolone and beta-lactam antimicrobial agents. 
Antimicrobial Agents and Chemotherapy  1990;34(10):1938-1943.
Norfloxacin, ofloxacin, and other new quinolones, which are antagonists of the enzyme DNA gyrase, rapidly kill bacteria by largely unknown mechanisms. Earlier, we isolated, after mutagenesis, Escherichia coli DS1, which exhibited reduced killing by quinolones. We evaluated the killing of DS1 and several other strains by quinolones and beta-lactams. In time-killing studies with norfloxacin, DS1 was killed 1 to 2 log10 units compared to 4 to 5 log10 units for the wild-type parent strain KL16, thus revealing that DS1 is a high-persistence (hip) mutant. DS1 exhibited a similar high-persistence pattern for the beta-lactam ampicillin and reduced killing by drugs that differed in their affinities for penicillin-binding proteins, including cefoxitin, cefsulodin, imipenem, mecillinam, and piperacillin. Conjugation and P1 transduction studies identified a novel mutant locus (termed hipQ) in the 2-min region of the DS1 chromosome necessary for reduced killing by norfloxacin and ampicillin. E. coli KL500, which was isolated for reduced killing by norfloxacin without mutagenesis, exhibited reduced killing by ampicillin. E. coli HM23, a hipA (34 min) mutant that was isolated earlier for reduced killing by ampicillin, also exhibited high persistence to norfloxacin. DS1 differed from HM23, however, in the map location of its hip mutation, lack of cold sensitivity, and reduced killing by coumermycin. Results of these studies with strains DS1, KL500, and HM23 demonstrate overlap in the pathways of killing of E. coli by quinolones and beta-lactams and identify hipQ, a new mutant locus that is involved in a high-persistence pattern of reduced killing by norfloxacin and ampicillin.
PMCID: PMC171968  PMID: 1963289
18.  Age of Inoculum Strongly Influences Persister Frequency and Can Mask Effects of Mutations Implicated in Altered Persistence ▿† 
Journal of Bacteriology  2011;193(14):3598-3605.
The majority of cells transferred from stationary-phase culture into fresh medium resume growth quickly, while a few remain in a nongrowing state for longer. These temporarily nonproliferating bacteria are tolerant of several bactericidal antibiotics and constitute a main source of persisters. Several genes have been shown to influence the frequency of persisters in Escherichia coli, although the exact mechanism underlying persister formation is unknown. This study demonstrates that the frequency of persisters is highly dependent on the age of the inoculum and the medium in which it has been grown. The hipA7 mutant had 1,000 times more persisters than the wild type when inocula were sampled from younger stationary-phase cultures. When started after a long stationary phase, the two displayed equal and elevated persister frequencies. The lower persister frequencies of glpD, dnaJ, and surA knockout strains were increased to the level of the wild type when inocula aged. The mqsR and phoU deletions showed decreased persister levels only when the inocula were from aged cultures, while sucB and ygfA deletions had decreased persister levels irrespective of the age of the inocula. A dependency on culture conditions underlines the notion that during screening for mutants with altered persister frequencies, the exact experimental details are of great importance. Unlike ampicillin and norfloxacin, which always leave a fraction of bacteria alive, amikacin killed all cells in the growth resumption experiment. It was concluded that the frequency of persisters depends on the conditions of inoculum cultivation, particularly its age, and the choice of antibiotic.
doi:10.1128/JB.00085-11
PMCID: PMC3133311  PMID: 21602347
19.  Delineation of a Bacterial Starvation Stress Response Network Which Can Mediate Antibiotic Tolerance Development▿  
This study aimed at elucidating the physiological basis of bacterial antibiotic tolerance. By use of a combined phenotypic and gene knockout approach, exogenous nutrient composition was identified as a crucial environmental factor which could mediate progressive development of tolerance with markedly varied drug specificity and sustainability. Deprivation of amino acids was a prerequisite for tolerance formation, conferring condition-specific phenotypes against inhibitors of cell wall synthesis and DNA replication (ampicillin and ofloxacin, respectively), according to the relative abundances of ammonium salts, phosphate, and nucleobases. Upon further depletion of glucose, this variable phase consistently evolved into a sustainable mode, along with enhanced capacity to withstand the effect of the protein synthesis inhibitor gentamicin. Nevertheless, all phenotypes produced during spontaneous nutrient depletion lacked the sustainable, multidrug-tolerant features exhibited by the stationary-phase population and were attributed to complex interaction between starvation-mediated metabolic and stress protection responses on the basis of the following reasons: (i) the nutrition-dependent tolerance characteristics observed suggested that adaptive biosynthetic mechanisms could suppress but not fully avert tolerance under transient starvation conditions; (ii) formation of specific phenotypes could be inhibited by suppressing protein synthesis prior to nutrient depletion; (iii) bacteriostatic drugs produced only weak tolerance in the absence of starvation signals; and (iv) the attenuation of the stringent and SOS responses, as well as the functionality of other putative tolerance determinants, including rpoS, hipA, glpD, and phoU, could alter the induction requirement and drug specificity of the resultant phenotypes. These data reveal the common physiological grounds characteristic of starvation responses and the onset of antibiotic tolerance in bacteria.
doi:10.1128/AAC.01218-09
PMCID: PMC2825962  PMID: 20086164
20.  Multiple antibiotic resistance (mar) locus protects Escherichia coli from rapid cell killing by fluoroquinolones. 
The multiple antibiotic resistance (mar) locus in Escherichia coli consists of two divergently expressed operons (marC and marRAB), both of which contribute to the Mar phenotype. Overexpression of the marRAB operon protected E. coli against rapid cell killing by fluoroquinolones. Inactivation of the operon in mar mutants restored a wild-type bactericidal susceptibility. Both operons of the locus were required for protection from the quinolone-mediated bactericidal activity in mar locus deletion mutants. The effect was lost at high concentrations of fluoroquinolones, unlike the case for the previously described genes hipA and hipQ. The inducible mar locus appears to specify a novel antibactericidal mechanism which may play a role in the emergence of fluoroquinolone-resistant clinical E. coli isolates.
PMCID: PMC163305  PMID: 8723480
21.  GlpD and PlsB Participate in Persister Cell Formation in Escherichia coli 
Journal of Bacteriology  2006;188(14):5136-5144.
Bacterial populations produce dormant persister cells that are resistant to killing by all antibiotics currently in use, a phenomenon known as multidrug tolerance (MDT). Persisters are phenotypic variants of the wild type and are largely responsible for MDT of biofilms and stationary populations. We recently showed that a hipBA toxin/antitoxin locus is part of the MDT mechanism in Escherichia coli. In an effort to find additional MDT genes, an E. coli expression library was selected for increased survival to ampicillin. A clone with increased persister production was isolated and was found to overexpress the gene for the conserved aerobic sn-glycerol-3-phosphate dehydrogenase GlpD. The GlpD overexpression strain showed increased tolerance to ampicillin and ofloxacin, while a strain with glpD deleted had a decreased level of persisters in the stationary state. This suggests that GlpD is a component of the MDT mechanism. Further genetic studies of mutants affected in pathways involved in sn-glycerol-3-phosphate metabolism have led to the identification of two additional multidrug tolerance loci, glpABC, the anaerobic sn-glycerol-3-phosphate dehydrogenase, and plsB, an sn-glycerol-3-phosphate acyltransferase.
doi:10.1128/JB.00369-06
PMCID: PMC1539972  PMID: 16816185
22.  The Chromosomal Toxin Gene yafQ Is a Determinant of Multidrug Tolerance for Escherichia coli Growing in a Biofilm▿  
Escherichia coli is refractory to elevated doses of antibiotics when it is growing in a biofilm, and this is potentially due to high numbers of multidrug-tolerant persister cells in the surface-adherent population. Previously, the chromosomal toxin-antitoxin loci hipBA and relBE have been linked to the frequency at which persister cells occur in E. coli populations. In the present study, we focused on the dinJ-yafQ-encoded toxin-antitoxin system and hypothesized that deletion of the toxin gene yafQ might influence cell survival in antibiotic-exposed biofilms. By using confocal laser scanning microscopy and viable cell counting, it was determined that a ΔyafQ mutant produced biofilms with a structure and a cell density equivalent to those of the parental strain. In-depth susceptibility testing identified that relative to wild-type E. coli, the ΔyafQ strain had up to a ∼2,400-fold decrease in cell survival after the biofilms were exposed to bactericidal concentrations of cefazolin or tobramycin. Corresponding to these data, controlled overexpression of yafQ from a high-copy-number plasmid resulted in up to a ∼10,000-fold increase in the number of biofilm cells surviving exposure to these bactericidal drugs. In contrast, neither the inactivation nor the overexpression of yafQ affected the tolerance of biofilms to doxycycline or rifampin (rifampicin). Furthermore, deletion of yafQ did not affect the tolerance of stationary-phase planktonic cells to any of the antibacterials tested. These results suggest that yafQ mediates the tolerance of E. coli biofilms to multiple but specific antibiotics; moreover, our data imply that this cellular pathway for persistence is likely different from that of multidrug-tolerant cells in stationary-phase planktonic cell cultures.
doi:10.1128/AAC.00043-09
PMCID: PMC2687228  PMID: 19307375
23.  Mycobacterium tuberculosis Hip1 Modulates Macrophage Responses through Proteolysis of GroEL2 
PLoS Pathogens  2014;10(5):e1004132.
Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses and persist within macrophages. We have previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, prevents robust macrophage activation and dampens host pro-inflammatory responses, allowing Mtb to delay immune detection and accelerate disease progression. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a direct substrate of Hip1 protease activity. Cleavage of GroEL2 is specifically inhibited by serine protease inhibitors. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during Mtb growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our studies point to Hip1-dependent proteolysis as a novel regulatory mechanism that helps Mtb respond rapidly to changing host immune environments during infection. These findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against Mtb.
Author Summary
Mycobacterium tuberculosis (Mtb) faces adverse conditions within host cells and has evolved many mechanisms to adapt quickly to the hostile immune environment. We have previously shown that an Mtb factor, Hip1, is important for Mtb virulence and for modulating host immunity. While Hip1 was predicted to be a protease, its enzymatic activity and molecular mechanism of function remained unclear. We have now characterized the biochemistry of Hip1 and conclusively show that Hip1 is a serine protease that can process peptide substrates. Further, we have identified a physiological target for Hip1 activity in Mtb, the Mtb chaperone-like protein GroEL2. Interestingly, cleavage of GroEL2 by Hip1 converted GroEL2 from a multimeric cell wall-associated protein to a monomeric form that is secreted extracellularly. Importantly, we show that cleavage of GroEL2 by Hip1 is biologically relevant and promotes dampening of macrophage responses during Mtb infection. Thus, our studies have uncovered a fine-tuned strategy of immune modulation at the protein level that involves regulating Hip1-GroEL2 interactions and provide key molecular insights for targeting Hip1 protease activity for inhibition.
doi:10.1371/journal.ppat.1004132
PMCID: PMC4022732  PMID: 24830429
24.  Modeling suggests that gene circuit architecture controls phenotypic variability in a bacterial persistence network 
BMC Systems Biology  2012;6:47.
Background
Bacterial persistence is a non-inherited bet-hedging mechanism where a subpopulation of cells enters a dormant state, allowing those cells to survive environmental stress such as treatment with antibiotics. Persister cells are not mutants; they are formed by natural stochastic variation in gene expression. Understanding how regulatory architecture influences the level of phenotypic variability can help us explain how the frequency of persistence events can be tuned.
Results
We present a model of the regulatory network controlling the HipBA toxin-antitoxin system from Escherichia coli. Using a biologically realistic model we first determine that the persistence phenotype is not the result of bistability within the network. Next, we develop a stochastic model and show that cells can enter persistence due to random fluctuations in transcription, translation, degradation, and complex formation. We then examine alternative gene circuit architectures for controlling hipBA expression and show that networks with more noise (more persisters) and less noise (fewer persisters) are straightforward to achieve. Thus, we propose that the gene circuit architecture can be used to tune the frequency of persistence, a trait that can be selected for by evolution.
Conclusions
We develop deterministic and stochastic models describing how the regulation of toxin and antitoxin expression influences phenotypic variation within a population. Persistence events are the result of stochastic fluctuations in toxin levels that cross a threshold, and their frequency is controlled by the regulatory topology governing gene expression.
doi:10.1186/1752-0509-6-47
PMCID: PMC3434061  PMID: 22607777
Persister; Toxin-antitoxin; Gene regulatory network; Feedback
25.  Role of Global Regulators and Nucleotide Metabolism in Antibiotic Tolerance in Escherichia coli▿  
Bacterial populations produce a small number of persister cells that exhibit multidrug tolerance. Persister cells are largely responsible for the antibiotic recalcitrance of biofilm infections. The mechanism of persister cell formation largely remains unknown due to the challenges in identifying persister genes. We screened an ordered comprehensive library of 3,985 Escherichia coli knockout strains to identify mutants with altered antibiotic tolerance. Stationary-state cultures in 96-well plates were exposed to ofloxacin at a concentration which allows only tolerant persister cells to survive. The persister cell level of each culture was determined. A total of 150 mutants with decreased persistence were identified in the initial screen, and subsequent validation confirmed that neither the growth rate nor the ofloxacin MIC was affected for 10 of them. The genes affected in these strains were dnaJ and dnaK (chaperones), apaH (diadenosine tetraphosphatase), surA (peptidyl-prolyl cis-trans isomerase), fis and hns (global regulators), hnr (response regulator of RpoS), dksA (transcriptional regulator of rRNA transcription), ygfA (5-formyl-tetrahydrofolate cyclo-ligase), and yigB (flavin mononucleotide [FMN] phosphatase). The prominent presence of global regulators among these strains pointed to the likely redundancy of persister cell formation mechanisms: the elimination of a regulator controlling several redundant persister genes would be expected to produce a phenotype. This observation is consistent with previous findings for a possible role of redundant genes such as toxin/antitoxin modules in persister cell formation. ygfA and yigB were of special interest. The mammalian homolog of YgfA (methenyltetrahydrofolate synthetase) catalyzes the conversion of 5-formyl-tetrahydrofolate (THF) into the rapidly degraded 5,10-methenyl-THF, depleting the folate pool. The YigB protein is a phosphatase of FMN which would deplete the pool of this cofactor. Stochastic overexpression of these genes could lead to dormancy and, hence, tolerance by depleting the folate and FMN pools, respectively. Consistent with this scenario, the overexpression of both genes produced increased tolerance to ofloxacin.
doi:10.1128/AAC.00144-08
PMCID: PMC2493092  PMID: 18519731

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