Satish Raina

Phosphorylation-mediated regulation of heat shock response in Escherichia coli

Escherichia coli has two heat shock regulons under the transcriptional control of Eσ32 and EσE RNA polymerases. These polymerases control the expression of genes, the products of which are needed for correct folding of proteins in the cytoplasm and the extracytoplasm respectively. In this study, we report that mutations in a tyrosine phosphatase‐encoding gene led to decreased activity of these heat shock regulons. The activity of the tyrosine phosphatase is presumably co‐ordinated with that of a cognate kinase. We show here that mutants deleted for the phosphatase‐encoding gene accumulate phosphorylated RpoH. We find that RpoH is phosphorylated at amino acid position 260, which is located in the conserved region 4.2, and that this phosphorylation event attenuates RpoH activity as a sigma factor. The rpoH Tyr‐260Ala mutation confers a temperature‐sensitive phenotype that leads to an altered heat shock response. Additionally, we show that the antisigma factor RseA is phosphorylated at the N‐terminally located Tyr‐38 and that this phosphorylation presumably alters its binding affinity towards σE.

Escherichia coli K-12 Suppressor-free Mutants Lacking Early Glycosyltransferases and Late Acyltransferases: minimal lipopolysaccharide structure and induction of envelope stress response.

To elucidate the minimal lipopolysaccharide (LPS) structure needed for the viability of Escherichia coli, suppressor-free strains lacking either the 3-deoxy-d-manno-oct-2-ulosonic acid transferase waaA gene or derivatives of the heptosyltransferase I waaC deletion with lack of one or all late acyltransferases (lpxL/M/P) and/or various outer membrane biogenesis factors were constructed. Δ(waaC lpxL lpxM lpxP) and waaA mutants exhibited highly attenuated growth, whereas simultaneous deletion of waaC and surA was lethal. Analyses of LPS of suppressor-free waaA mutants grown at 21 °C, besides showing accumulation of free lipid IVA precursor, also revealed the presence of its pentaacylated and hexaacylated derivatives, indicating in vivo late acylation can occur without Kdo. In contrast, LPS of Δ(waaC lpxL lpxM lpxP) strains showed primarily Kdo2-lipid IVA, indicating that these minimal LPS structures are sufficient to support growth of E. coli under slow-growth conditions at 21/23 °C. These lipid IVA derivatives could be modified biosynthetically by phosphoethanolamine, but not by 4-amino-4-deoxy-l-arabinose, indicating export defects of such minimal LPS. ΔwaaA and Δ(waaC lpxL lpxM lpxP) exhibited cell-division defects with a decrease in the levels of FtsZ and OMP-folding factor PpiD. These mutations led to strong constitutive additive induction of envelope responsive CpxR/A and σE signal transduction pathways. Δ(lpxL lpxM lpxP) mutant, with intact waaC, synthesized tetraacylated lipid A and constitutively incorporated a third Kdo in growth medium inducing synthesis of P-EtN and l-Ara4N. Overexpression of msbA restored growth of Δ(lpxL lpxM lpxP) under fast-growing conditions, but only partially that of the Δ(waaC lpxL lpxM lpxP) mutant. This suppression could be alleviated by overexpression of certain mutant msbA alleles or the single-copy chromosomal MsbA-498V variant in the vicinity of Walker-box II.

Molecular Strategy for Survival at a Critical High Temperature in Eschierichia coli

The molecular mechanism supporting survival at a critical high temperature (CHT) in Escherichia coli was investigated. Genome-wide screening with a single-gene knockout library provided a list of genes indispensable for growth at 47°C, called thermotolerant genes. Genes for which expression was affected by exposure to CHT were identified by DNA chip analysis. Unexpectedly, the former contents did not overlap with the latter except for dnaJ and dnaK, indicating that a specific set of non-heat shock genes is required for the organism to survive under such a severe condition. More than half of the mutants of the thermotolerant genes were found to be sensitive to H2O2 at 30°C, suggesting that the mechanism of thermotolerance partially overlaps with that of oxidative stress resistance. Their encoded enzymes or proteins are related to outer membrane organization, DNA double-strand break repair, tRNA modification, protein quality control, translation control or cell division. DNA chip analyses of essential genes suggest that many of the genes encoding ribosomal proteins are down-regulated at CHT. Bioinformatics analysis and comparison with the genomic information of other microbes suggest that E. coli possesses several systems for survival at CHT. This analysis allows us to speculate that a lipopolysaccharide biosynthesis system for outer membrane organization and a sulfur-relay system for tRNA modification have been acquired by horizontal gene transfer.

Dissection of σE-dependent cell lysis in Escherichia coli: roles of RpoE regulators RseA, RseB and periplasmic folding catalyst PpiD

To understand the mechanism of σE‐dependent cell lysis, we examined the consequences of deletion derivatives of rpoE regulators rseA, rseB and rseC on σE transcription, on levels of free versus membrane‐bound σE and on OMP‐biogenesis limiting factor(s) that could impact cell lysis. RT‐PCR showed that individual nonpolar ΔrseA and ΔrseB increased the rpoE expression to varying extents, with pronounced induction in ΔrseA. Significantly the ratio of soluble (free) versus membrane‐bound form of RpoE increased in ΔrseA, however without increase of its total amount, unraveling furthermore complexity in RpoE regulation. Significant characteristics of cell lysis, accompanied by a severe reduction in the levels of periplasmic OMP‐folding factor (PpiD), were observed in ΔrseA. The cell‐lysis phenotype of ΔrseA was suppressed by either rseA or ppiD plasmids, but neither by rseB nor by rseC clones. However, the cell lysis of the wild‐type strain was almost completely repressed not only by the rseA clone but also by the rseB clone, suggesting RseB might be limiting in vivo. Thus, increase in the ratio of free σE in rseA mutants with a concomitant reduction in PpiD levels can account for σE‐dependent lysis in concert with a potential role of small RNAs on the lysis process.

Regulated Control of the Assembly and Diversity of LPS by Noncoding sRNAs

The outer membrane (OM) of Gram-negative bacteria is asymmetric due to the presence of lipopolysaccharide (LPS) facing the outer leaflet of the OM and phospholipids facing the periplasmic side. LPS is essential for bacterial viability, since it provides a permeability barrier and is a major virulence determinant in pathogenic bacteria. In Escherichia coli, several steps of LPS biosynthesis and assembly are regulated by the RpoE sigma factor and stress responsive two-component systems as well as dedicated small RNAs. LPS composition is highly heterogeneous and dynamically altered upon stress and other challenges in the environment because of the transcriptional activation of RpoE regulon members and posttranslational control by RpoE-regulated Hfq-dependent RybB and MicA sRNAs. The PhoP/Q two-component system further regulates Kdo2-lipid A modification via MgrR sRNA. Some of these structural alterations are critical for antibiotic resistance, OM integrity, virulence, survival in host, and adaptation to specific environmental niches. The heterogeneity arises following the incorporation of nonstoichiometric modifications in the lipid A part and alterations in the composition of inner and outer core of LPS. The biosynthesis of LPS and phospholipids is tightly coupled. This requires the availability of metabolic precursors, whose accumulation is controlled by sRNAs like SlrA, GlmZ, and GlmY.

Fatty Acyl Benzamido Antibacterials Based on Inhibition of DnaK-catalyzed Protein Folding

We have reported that the hsp70 chaperone DnaK from Escherichia coli might assist protein folding by catalyzing the cis/trans isomerization of secondary amide peptide bonds in unfolded or partially folded proteins. In this study a series of fatty acylated benzamido inhibitors of the cis/trans isomerase activity of DnaK was developed and tested for antibacterial effects in E. coli MC4100 cells. Nα-[Tetradecanoyl-(4-aminomethylbenzoyl)]-l-asparagine is the most effective antibacterial with a minimal inhibitory concentration of 100 ± 20 μg/ml. The compounds were shown to compete with fluorophore-labeled σ32-derived peptide for the peptide binding site of DnaK and to increase the fraction of aggregated proteins in heat-shocked bacteria. Despite its inability to serve as a folding helper in vivo a DnaK-inhibitor complex was still able to sequester an unfolded protein in vitro. Structure activity relationships revealed a distinct dependence of DnaK-assisted refolding of luciferase on the fatty acyl chain length, whereas the minimal inhibitory concentration was most sensitive to the structural nature of the benzamido core. We conclude that the isomerase activity of DnaK is a major survival factor in the heat shock response of bacteria and that small molecule inhibitors can lead to functional inactivation of DnaK and thus will display antibacterial activity.

Assembly of Lipopolysaccharide in Escherichia coli Requires the Essential LapB Heat Shock Protein

Background: The mechanism of coordination between LPS synthesis and translocation is unknown. Results: Two new proteins, LapA and LapB, co-purify with LPS transport proteins. lapB mutants display defects in lipid A and core assembly. Conclusion: lapB mutants accumulate precursor LPS core species and exhibit elevated levels of LpxC. Significance: Coordinated assembly of LPS is a critical step for targeting to the outer membrane. Here, we describe two new heat shock proteins involved in the assembly of LPS in Escherichia coli, LapA and LapB (lipopolysaccharide assembly protein A and B). lapB mutants were identified based on an increased envelope stress response. Envelope stress-responsive pathways control key steps in LPS biogenesis and respond to defects in the LPS assembly. Accordingly, the LPS content in ΔlapB or Δ(lapA lapB) mutants was elevated, with an enrichment of LPS derivatives with truncations in the core region, some of which were pentaacylated and exhibited carbon chain polymorphism. Further, the levels of LpxC, the enzyme that catalyzes the first committed step of lipid A synthesis, were highly elevated in the Δ(lapA lapB) mutant. Δ(lapA lapB) mutant accumulated extragenic suppressors that mapped either to lpxC, waaC, and gmhA, or to the waaQ operon (LPS biosynthesis) and lpp (Brauns lipoprotein). Increased synthesis of either FabZ (3-R-hydroxymyristoyl acyl carrier protein dehydratase), slrA (novel RpoE-regulated non-coding sRNA), lipoprotein YceK, toxin HicA, or MurA (UDP-N-acetylglucosamine 1-carboxyvinyltransferase) suppressed some of the Δ(lapA lapB) defects. LapB contains six tetratricopeptide repeats and, at the C-terminal end, a rubredoxin-like domain that was found to be essential for its activity. In pull-down experiments, LapA and LapB co-purified with LPS, Lpt proteins, FtsH (protease), DnaK, and DnaJ (chaperones). A specific interaction was also observed between WaaC and LapB. Our data suggest that LapB coordinates assembly of proteins involved in LPS synthesis at the plasma membrane and regulates turnover of LpxC, thereby ensuring balanced biosynthesis of LPS and phospholipids consistent with its essentiality.

Molecular Basis of Lipopolysaccharide Heterogeneity in Escherichia coli ENVELOPE STRESS-RESPONSIVE REGULATORS CONTROL THE INCORPORATION OF GLYCOFORMS WITH A THIRD 3-DEOXY-α-d-MANNO-OCT-2-ULOSONIC ACID AND RHAMNOSE

Background: LPS is essential for viability, although it is highly heterogeneous. Results: Synthesis of different glycoforms is regulated by differential expression of WaaZ (KdoIII transferase) and WaaR (glycosyltransferase). RpoE-transcribed rybB sRNA represses WaaR synthesis, and ppGpp is required for KdoIII incorporation in RpoE-inducing conditions. Conclusion: RpoE induction causes truncation of outer core and rhamnose addition to KdoIII. Significance: LPS alterations are crucial for outer membrane function. Mass spectrometric analyses of lipopolysaccharide (LPS) from isogenic Escherichia coli strains with nonpolar mutations in the waa locus or overexpression of their cognate genes revealed that waaZ and waaS are the structural genes required for the incorporation of the third 3-deoxy-α-d-manno-oct-2-ulosonic acid (Kdo) linked to Kdo disaccharide and rhamnose, respectively. The incorporation of rhamnose requires prior sequential incorporation of the Kdo trisaccharide. The minimal in vivo lipid A-anchored core structure Kdo2Hep2Hex2P1 in the LPS from ΔwaaO (lacking α-1,3-glucosyltransferase) could incorporate Kdo3Rha, without the overexpression of the waaZ and waaS genes. Examination of LPS heterogeneity revealed overlapping control by RpoE σ factor, two-component systems (BasS/R and PhoB/R), and ppGpp. Deletion of RpoE-specific anti-σ factor rseA led to near-exclusive incorporation of glycoforms with the third Kdo linked to Kdo disaccharide. This was accompanied by concomitant incorporation of rhamnose, linked to either the terminal third Kdo or to the second Kdo, depending upon the presence or absence of phosphoethanolamine on the second Kdo with truncation of the outer core. This truncation in ΔrseA was ascribed to decreased levels of WaaR glycosyltransferase, which was restored to wild-type levels, including overall LPS composition, upon the introduction of rybB sRNA deletion. Thus, ΔwaaR contained LPS primarily with Kdo3 without any requirement for lipid A modifications. Accumulation of a glycoform with Kdo3 and 4-amino-4-deoxy-l-arabinose in lipid A in ΔrseA required ppGpp, being abolished in a Δ(ppGpp0 rseA). Furthermore, Δ(waaZ lpxLMP) synthesizing tetraacylated lipid A exhibited synthetic lethality at 21–23°C pointing to the significance of the incorporation of the third Kdo.

Molecular and Structural Basis of Inner Core Lipopolysaccharide Alterations in Escherichia coli INCORPORATION OF GLUCURONIC ACID AND PHOSPHOETHANOLAMINE IN THE HEPTOSE REGION

Background: Some of the enzymes that are required for LPS modification(s) are unknown. Results: LPS modifications involving addition of glucuronic acid to heptose III and phosphoethanolamine transfer to heptose I require products of two new genes waaH and eptC, respectively. Conclusion: Glucuronic acid addition requires PhoB/R activation, and phosphoethanolamine transfer confers detergent resistance. Significance: Nonstoichiometric LPS alterations reflect LPS structural flexibility in response to stress conditions. It is well established that lipopolysaccharide (LPS) often carries nonstoichiometric substitutions in lipid A and in the inner core. In this work, the molecular basis of inner core alterations and their physiological significance are addressed. A new inner core modification of LPS is described, which arises due to the addition of glucuronic acid on the third heptose with a concomitant loss of phosphate on the second heptose. This was shown by chemical and structural analyses. Furthermore, the gene whose product is responsible for the addition of this sugar was identified in all Escherichia coli core types and in Salmonella and was designated waaH. Its deduced amino acid sequence exhibits homology to glycosyltransferase family 2. The transcription of the waaH gene is positively regulated by the PhoB/R two-component system in a growth phase-dependent manner, which is coordinated with the transcription of the ugd gene explaining the genetic basis of this modification. Glucuronic acid modification was observed in E. coli B, K12, R2, and R4 core types and in Salmonella. We also show that the phosphoethanolamine (P-EtN) addition on heptose I in E. coli K12 requires the product of the ORF yijP, a new gene designated as eptC. Incorporation of P-EtN is also positively regulated by PhoB/R, although it can occur at a basal level without a requirement for any regulatory inducible systems. This P-EtN modification is essential for resistance to a variety of factors, which destabilize the outer membrane like the addition of SDS or challenge to sublethal concentrations of Zn2+.

Multiple Transcriptional Factors Regulate Transcription of the rpoE Gene in Escherichia coli under Different Growth Conditions and When the Lipopolysaccharide Biosynthesis Is Defective.

The RpoE σ factor is essential for the viability of Escherichia coli. RpoE regulates extracytoplasmic functions including lipopolysaccharide (LPS) translocation and some of its non-stoichiometric modifications. Transcription of the rpoE gene is positively autoregulated by EσE and by unknown mechanisms that control the expression of its distally located promoter(s). Mapping of 5′ ends of rpoE mRNA identified five new transcriptional initiation sites (P1 to P5) located distal to EσE-regulated promoter. These promoters are activated in response to unique signals. Of these P2, P3, and P4 defined major promoters, recognized by RpoN, RpoD, and RpoS σ factors, respectively. Isolation of trans-acting factors, in vitro transcriptional and gel retardation assays revealed that the RpoN-recognized P2 promoter is positively regulated by a QseE/F two-component system and NtrC activator, whereas the RpoD-regulated P3 promoter is positively regulated by a Rcs system in response to defects in LPS core biosynthesis, overproduction of certain lipoproteins, and the global regulator CRP. Strains synthesizing Kdo2-LA LPS caused up to 7-fold increase in the rpoEP3 activity, which was abrogated in Δ(waaC rcsB). Overexpression of a novel 73-nucleotide sRNA rirA (RfaH interacting RNA) generated by the processing of 5′ UTR of the waaQ mRNA induces the rpoEP3 promoter activity concomitant with a decrease in LPS content and defects in the O-antigen incorporation. In the presence of RNA polymerase, RirA binds LPS regulator RfaH known to prevent premature transcriptional termination of waaQ and rfb operons. RirA in excess could titrate out RfaH causing LPS defects and the activation of rpoE transcription.