Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p)ppGpp synthesis

The ability of a high frequency (10(-2)) of Escherichia coli to survive prolonged exposure to penicillin antibiotics, called high persistence, is associated with mutations in the hipA gene. The hip operon is located in the chromosomal terminus near dif and consists of two genes, hipA and hipB. The wild-type hipA gene encodes a toxin, whereas hipB encodes a DNA-binding protein that autoregulates expression of the hip operon and binds to HipA to nullify its toxic effects. We have characterized the hipA7 allele, which confers high persistence, and established that HipA7 is non-toxic, contains two mutations (G22S and D291A) and that both mutations are required for the full range of phenotypes associated with hip mutants. Furthermore, expression of hipA7 in the absence of hipB is sufficient to establish the high persistent phenotype, indicating that hipB is not required. There is a strong correlation between the frequency of persister cells generated by hipA7 strains and cell density, with hipA7 strains generating a 20-fold higher frequency of persisters as cultures approach stationary phase. It is also demonstrated that relA knock-outs diminish the high persistent phenotype in hipA7 mutants and that relA spoT knock-outs eliminate high persistence altogether, suggesting that hipA7 facilitates the establishment of the persister state by inducing (p)ppGpp synthesis. Consistent with this proposal, ectopic expression of relA' from a plasmid was shown to increase the number of persistent cells produced by hipA7 relA double mutants by 100-fold or more. A model is presented that postulates that hipA7 increases the basal level of (p)ppGpp synthesis, allowing a significantly greater percentage of cells in a population to assume a persistent, antibiotic-insensitive state by potentiating a rapid transition to a dormant state upon application of stress.     

Peptidoglycan Synthesis in Cocci and Rods of a pH-Dependent, Morphologically Conditional Mutant of Klebsiella pneumoniae

Mir M7 is a spontaneous morphologically conditional mutant of Klebsiella pneumoniae which grows as round cells (cocci) at pH 7 and as normal rods at pH 5.8. We studied the rates of peptidoglycan synthesis of cocci and rods growing at pH values of 7 and 5.8, respectively. It was found that exponentially growing cocci produced a reduced amount of peptidoglycan per cell, compared with rods. Moreover, a shift of cocci to the permissive pH (5.8) caused an increase in the rate of peptidoglycan synthesis, whereas the reverse shift of rods to pH 7 determined a twofold reduction in the rate of [(3)H]diaminopimelic acid incorporation. During synchronous growth at pH 7, the rate of peptidoglycan synthesis after cell division decreased with time and rose before and during the first division. The susceptibilities of rods and cocci to beta-lactam antibiotics were also studied. It was found that cocci were more sensitive both to penicillin G and to cephalexin than were rods, but they showed a high level of resistance to mecillinam. The peculiar behavior of this mutant was interpreted as supporting the existence in bacterial rods of two different sites for peptidoglycan synthesis: one responsible for lateral wall elongation and one responsible for septum formation. In Mir M7, shape damage is described as dependent on the specific inhibition, at the nonpermissive pH, of the site for lateral wall extension.     

Ectopic overexpression of wild-type and mutant hipA genes in Escherichia coli: effects on macromolecular synthesis and persister formation

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.     

Unstable Escherichia coli L forms revisited: growth requires peptidoglycan synthesis

Growing bacterial L forms are reputed to lack peptidoglycan, although cell division is normally inseparable from septal peptidoglycan synthesis. To explore which cell division functions L forms use, we established a protocol for quantitatively converting a culture of a wild-type Escherichia coli K-12 strain overnight to a growing L-form-like state by use of the beta-lactam cefsulodin, a specific inhibitor of penicillin-binding proteins (PBPs) 1A and 1B. In rich hypertonic medium containing cefsulodin, all cells are spherical and osmosensitive, like classical L forms. Surprisingly, however, mutant studies showed that colony formation requires d-glutamate, diaminopimelate, and MurA activity, all of which are specific to peptidoglycan synthesis. High-performance liquid chromatography analysis confirmed that these L-form-like cells contain peptidoglycan, with 7% of the normal amount. Moreover, the beta-lactam piperacillin, a specific inhibitor of the cell division protein PBP 3, rapidly blocks the cell division of these L-form-like cells. Similarly, penicillin-induced L-form-like cells, which grow only within the agar layers of rich hypertonic plates, also require d-glutamate, diaminopimelate, and MurA activity. These results strongly suggest that cefsulodin- and penicillin-induced L-form-like cells of E. coli-and possibly all L forms-have residual peptidoglycan synthesis which is essential for their growth, probably being required for cell division.     

Genomic channeling in bacterial cell division

The bacterial dcw cluster is a group of genes involved in cell division and peptidoglycan synthesis. Comparison of the cluster across several bacterial genomes shows that its gene content and its gene order are conserved in distant bacterial lineages and, moreover, that, being most conserved in rod-shaped bacteria, the degree of conservation relates to bacterial morphology. We propose a model in which the selective pressure to maintain the cluster arises from the need to efficiently coordinate the processes of elongation and septation in rod-shaped bacteria. Gene order in the dcw cluster would be conserved as a result of mechanisms comprising: (i) a limited amount of peptidoglycan precursors required both for septation and elongation of the wall; (ii) co-translational assembly of the protein complexes involved in cell division and in the synthesis of the peptidoglycan precursors; and (iii) alternation in the cellular localization of the assembled complexes to participate either in the synthesis of the septal peptidoglycan and division, or in the synthesis of the lateral wall. The name genomic channeling is proposed for this model as it involves a genomic arrangement that could facilitate the assembly of specific protein complexes and their subsequent conveyance to specific locations in the crowded cytoplasm and the envelope.     

Peptidoglycan recognition proteins kill bacteria by activating protein-sensing two-component systems

Mammalian peptidoglycan recognition proteins (PGRPs), similar to antimicrobial lectins, bind the bacterial cell wall and kill bacteria through an unknown mechanism. We show that PGRPs enter the Gram-positive cell wall at the site of daughter cell separation during cell division. In Bacillus subtilis, PGRPs activate the CssR-CssS two-component system that detects and disposes of misfolded proteins that are usually exported out of bacterial cells. This activation results in membrane depolarization, cessation of intracellular peptidoglycan, protein, RNA and DNA synthesis, and production of hydroxyl radicals, which are responsible for bacterial death. PGRPs also bind the outer membrane of Escherichia coli and activate the functionally homologous CpxA-CpxR two-component system, which kills the bacteria. We exclude other potential bactericidal mechanisms, including inhibition of extracellular peptidoglycan synthesis, hydrolysis of peptidoglycan and membrane permeabilization. Thus, we reveal a previously unknown mechanism by which innate immunity proteins that bind the cell wall or outer membrane exploit the bacterial stress defense response to kill bacteria.     

hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis.

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.     

Cell division is antagonized by the activity of peptidoglycan endopeptidases that promote cell elongation

A peptidoglycan (PG) cell wall composed of glycans crosslinked by short peptides surrounds most bacteria and protects them against osmotic rupture. In Escherichia coli, cell elongation requires crosslink cleavage by PG endopeptidases to make space for the incorporation of new PG material throughout the cell cylinder. Cell division, on the contrary, requires the localized synthesis and remodeling of new PG at midcell by the divisome. Little is known about the factors that modulate transitions between these two modes of PG biogenesis. In a transposon-insertion sequencing screen to identify mutants synthetically lethal with a defect in the division protein FtsP, we discovered that mutants impaired for cell division are sensitive to elevated activity of the endopeptidases. Increased endopeptidase activity in these cells was shown to interfere with the assembly of mature divisomes, and conversely, inactivation of MepS was found to suppress the lethality of mutations in essential division genes. Overall, our results are consistent with a model in which the cell elongation and division systems are in competition with one another and that control of PG endopeptidase activity represents an important point of regulation influencing the transition from elongation to the division mode of PG biogenesis.     

Inhibition of cell wall synthesis and acylation of the penicillin binding proteins during prolonged exposure of growing Streptococcus pneumoniae to benzylpenicillin

Growing cultures of an autolysis-defective pneumococcal mutant were exposed to [3H]benzylpenicillin at various multiples of the minimal inhibitory concentration and incubated until the growth of the cultures was halted. During the process of growth inhibition, we determined the rates and degree of acylation of the five penicillin-binding proteins (PBPs) and the rates of peptidoglycan incorporation, protein synthesis, and turbidity increase. The time required for the onset of the inhibitory effects of benzylpenicillin was inversely related to the concentration of the antibiotic, and inhibition of peptidoglycan incorporation always preceded inhibition of protein synthesis and growth. When cultures first started to show the onset of growth inhibition, the same characteristic fraction of each PBP was in the acylated form in all cases, irrespective of the antibiotic concentration. Apparently, saturation of one or more PBPs with the antibiotic beyond these threshold levels is needed to bring about interference with normal peptidoglycan production and cellular growth. Although it was not possible to correlate the inhibition of cell wall synthesis or cell growth with the degree of acylation (percentage saturation) of any single PBP, there was a correlation between the amount of peptidoglycan synthesized and the actual amount of PBP 2b that was not acylated. In cultures exposed to benzylpenicillin concentrations greater than eight times the minimal inhibitory concentration, the rates of peptidoglycan incorporation underwent a rapid decline when bacterial growth stopped. However, in cultures exposed to lower concentrations of benzylpenicillin (one to six times the minimal inhibitory concentration) peptidoglycan synthesis continued at constant rate for prolonged periods, after the turbidity had ceased to increase. We conclude that inhibition of bacterial growth does not require a complete inhibition or even a major decline in the rate of peptidoglycan incorporation. Rather, inhibition of growth must be caused by an as yet undefined process that stops cell division when the rate of incorporation of peptidoglycan (or synthesis of protein) falls below a critical value.     

Control of cell shape and elongation by the rodA gene in Bacillus subtilis

The Escherichia coli rodA and ftsW genes and the spoVE gene of Bacillus subtilis encode membrane proteins that control peptidoglycan synthesis during cellular elongation, division and sporulation respectively. While rodA and ftsW are essential genes in E. coli , the B. subtilis spoVE gene is dispensable for growth and is only required for the synthesis of the spore cortex peptidoglycan. In this work, we report on the characterization of a B. subtilis gene, designated rodA , encoding a homologue of E. coli RodA. We found that the growth of a B. subtilis strain carrying a fusion of rodA to the IPTG-inducible P_spac promoter is inducer dependent. Limiting concentrations of inducer caused the formation of spherical cells, which eventually lysed. An increase in the level of IPTG induced a sphere-to-short rod transition that re-established viability. Higher levels of inducer restored normal cell length. Staining of the septal or polar cap peptidoglycan by a fluorescent lectin was unaffected during growth of the mutant under restrictive conditions. Our results suggest that rodA functions in maintaining the rod shape of the cell and that this function is essential for viability. In addition, RodA has an irreplaceable role in the extension of the lateral walls of the cell. Electron microscopy observations support these conclusions. The ultrastructural analysis further suggests that the growth arrest that accompanies loss of the rod shape is caused by the cell's inability to construct a division septum capable of spanning the enlarged cell. RodA is similar over its entire length to members of a large protein family (SEDS, for shape, elongation, division and sporulation). Members of the SEDS family are probably present in all eubacteria that synthesize peptidoglycan as part of their cell envelope.     

Spore cortex formation in Bacillus subtilis is regulated by accumulation of peptidoglycan precursors under the control of sigma K

The bacterial endospore cortex peptidoglycan is synthesized between the double membranes of the developing forespore and is required for attainment of spore dehydration and dormancy. The Bacillus subtilis spoVB, spoVD and spoVE gene products are expressed in the mother cell compartment early during sporulation and play roles in cortex synthesis. Here we show that mutations in these genes block synthesis of cortex peptidoglycan and cause accumulation of peptidoglycan precursors, indicating a defect at the earliest steps of peptidoglycan polymerization. Loss of spoIV gene products involved in activation of later, sigma(K)-dependent mother cell gene expression results in decreased synthesis of cortex peptidoglycan, even in the presence of the SpoV proteins that were synthesized earlier, apparently due to decreased precursor production. Data show that activation of sigma(K) is required for increased synthesis of the soluble peptidoglycan precursors, and Western blot analyses show that increases in the precursor synthesis enzymes MurAA, MurB, MurC and MurF are dependent on sigma(K) activation. Overall, our results indicate that a decrease in peptidoglycan precursor synthesis during early sporulation, followed by renewed precursor synthesis upon sigma(K) activation, serves as a regulatory mechanism for the timing of spore cortex synthesis.     

The rate and topography of cell wall synthesis during the division cycle of Escherichia coli using N-acetylglucosamine as a peptidoglycan label

The rates of synthesis of peptidoglycan and protein during the division cycle of Escherichia coli were measured by the membrane elution technique using cells differentially labelled with N-acetylglucosamine and leucine. During the first part of the division cycle the ratio of the rates of protein and peptidoglycan synthesis was constant. The rate of peptidoglycan synthesis, relative to the rate of protein synthesis, increased during the latter part of the division cycle. These results support a simple, bipartite model of cell surface increase in rod-shaped cells. Prior to the start of constriction the cell surface increases only by lateral wall extension. After cell constriction starts, the cell surface increases by both lateral wall and pole growth. The increase in surface area is partitioned between the lateral wall and the pole so that the volume of the cell increases exponentially. No variation in cell density occurs, because the increase in surface allows a continuous exponential increase in cell volume that accommodates the exponential increase in cell mass. The results are consistent with the constant density of the growing cell and the surface stress model for the regulation of cell surface synthesis. In addition, the elution pattern suggests that the membrane elution method does work by having the cells effectively bound to the membrane by their poles.     

Evidence for a peptidoglycan‐like structure in Orientia tsutsugamushi

Bacterial cell walls are composed of the large cross‐linked macromolecule peptidoglycan, which maintains cell shape and is responsible for resisting osmotic stresses. This is a highly conserved structure and the target of numerous antibiotics. Obligate intracellular bacteria are an unusual group of organisms that have evolved to replicate exclusively within the cytoplasm or vacuole of a eukaryotic cell. They tend to have reduced amounts of peptidoglycan, likely due to the fact that their growth and division takes place within an osmotically protected environment, and also due to a drive to reduce activation of the host immune response. Of the two major groups of obligate intracellular bacteria, the cell wall has been much more extensively studied in the Chlamydiales than the Rickettsiales. Here, we present the first detailed analysis of the cell envelope of an important but neglected member of the Rickettsiales, Orientia tsutsugamushi. This bacterium was previously reported to completely lack peptidoglycan, but here we present evidence supporting the existence of a peptidoglycan‐like structure in Orientia, as well as an outer membrane containing a network of cross‐linked proteins, which together confer cell envelope stability. We find striking similarities to the unrelated Chlamydiales, suggesting convergent adaptation to an obligate intracellular lifestyle.     

Peptidoglycan in obligate intracellular bacteria

Peptidoglycan is the predominant stress‐bearing structure in the cell envelope of most bacteria, and also a potent stimulator of the eukaryotic immune system. Obligate intracellular bacteria replicate exclusively within the interior of living cells, an osmotically protected niche. Under these conditions peptidoglycan is not necessarily needed to maintain the integrity of the bacterial cell. Moreover, the presence of peptidoglycan puts bacteria at risk of detection and destruction by host peptidoglycan recognition factors and downstream effectors. This has resulted in a selective pressure and opportunity to reduce the levels of peptidoglycan. In this review we have analysed the occurrence of genes involved in peptidoglycan metabolism across the major obligate intracellular bacterial species. From this comparative analysis, we have identified a group of predicted ‘peptidoglycan‐intermediate’ organisms that includes the Chlamydiae, Orientia tsutsugamushi, Wolbachia and Anaplasma marginale. This grouping is likely to reflect biological differences in their infection cycle compared with peptidoglycan‐negative obligate intracellular bacteria such as Ehrlichia and Anaplasma phagocytophilum, as well as obligate intracellular bacteria with classical peptidoglycan such as Coxiella, Buchnera and members of the Rickettsia genus. The signature gene set of the peptidoglycan‐intermediate group reveals insights into minimal enzymatic requirements for building a peptidoglycan‐like sacculus and/or division septum.     

Mutational dissection of the S/T-kinase StkP reveals crucial roles in cell division of Streptococcus pneumoniae

Eukaryotic-like serine/threonine-kinases are involved in the regulation of a variety of physiological processes in bacteria. In Streptococcus pneumoniae, deletion of the single serine/threonine-kinase gene stkP results in an aberrant cell morphology suggesting that StkP participates in pneumococcus cell division. To understand the function of StkP, we have engineered various pneumococcus strains expressing truncated or kinase-dead forms of StkP. We show that StkP kinase activity, but also its extracellular and cytoplasmic domains per se, are required for pneumococcus cell division. Indeed, we observe that mutant cells show round or elongated shapes with non-functional septa and a chain phenotype, delocalized sites of peptidoglycan synthesis and diffused membrane StkP localization. To gain understanding of the underlying StkP-mediated regulatory mechanism, we show that StkP specifically phosphorylates in vivo the cell division protein DivIVA on threonine 201. Pneumococcus cells expressing non-phosphorylatable DivIVA-T201A possess an elongated shape with a polar bulge and aberrant spatial organization of nascent peptidoglycan. This brings the first evidence of the importance of StkP in relationship to the phosphorylation of one of its substrates in cell division. It is concluded that StkP is a multifunctional protein that plays crucial functions in pneumococcus cell shape and division.     

Comparison of the nucleotide sequences of a yeast gene family. I. Distribution and spectrum of spontaneous base substitutions

The nucleotide sequences of closely related members of a gene family can be used to investigate spontaneous mutations. Here we analyse the sequences of different yeast invertase genes which are more than 93% identical in the coding region and share some very similar, but not identical sequences in the noncoding flanking regions. Since all except one of the invertase genes are active, most of the base substitutions are silent. Within the coding region the base substitutions are unevenly distributed, indicating that parts of the genes were homogenized, probably via gene conversion. Transitions occurred more frequently than transversions in both, coding and noncoding regions. In the coding region pyrimidine transitions were the most abundant event due to silent changes mainly in the third codon position. In the noncoding region pyrimidine and purine transitions were found at equal frequencies. Transversions inverting base pairs (A-T and G-C) outnumber transversions changing base pairs (A-C and G-T). While the spectrum of mutations in the coding region is influenced by selective pressure to maintain the amino acid sequence, the spectrum in the noncoding region may be much less affected by selective pressure.     

Relative affinity of vancomycin and ristocetin for cell walls and uridine diphosphate-N-acetylmuramyl pentapeptide

A determination of the relative affinity of vancomycin and ristocetin for isolated cell walls and for a peptidoglycan precursor was made. These antibiotics had previously been shown to adsorb to cell walls and to complex with peptides containing a d-alanyl-d-alanine C-terminus. By using (14)C-uridine diphosphate (UDP)-N-acetylmuramyl pentapeptide, it was shown that the complex which is formed between this peptidoglycan precursor and either vancomycin or ristocetin does not preclude adsorption of the antibiotics to cell walls of Micrococcus lysodeikticus. Complex formation between ristocetin and UDP-N-acetylmuramyl pentapeptide was assured by differential absorption spectra. However, when the complex was mixed with cell walls, the antibiotic was sedimented with the walls, and the radioactivity remained in the supernatant solution. This indication that ristocetin and vancomycin have a greater affinity for walls than for UDP-N-acetylmuramyl pentapeptide and that the complex per se does not bind to cell walls suggests that adsorption of these antibiotics to cell walls is probably responsible for the inhibition of peptidoglycan synthesis. This proposal is strengthened by the observation that complexed antibiotic is no less inhibitory for growth of Bacillus subtilis than free vancomycin or ristocetin.