Hyperexpression of the osmB gene in an acidic phospholipid-deficient Escherichia coli mutant

An Escherichia coli pgsA null mutant deficient in acidic phospholipids shows a thermosensitive cell lysis phenotype because of activation of the Rcs phosphorelay signal transduction system. We conducted a DNA microarray analysis with special attention to the genes affected by growth temperature in the mutant deficient in acidic phospholipids. Among the genes identified as highly expressed at high temperature in the pgsA null mutant, the osmB gene was shown to be dependent on the Rcs system for the high expression by dot blot hybridization. Induction of the cloned osmB in the pgsA null mutant caused the thermosensitive defect even in the absence of the Rcs system. Although the deletion of osmB did not suppress the thermosensitivity in the presence of the Rcs system, indicating a multifactorial nature of the deleterious effect of the Rcs activation, we suggest that the osmB hyperexpression is one of the causes of the Rcs-dependent lysis phenotype of the pgsA null mutant.     

RcsA-dependent and -independent growth defects caused by the activated Rcs phosphorelay system in the Escherichia coli pgsA null mutant

In the Escherichia coli pgsA null mutant, which lacks the major acidic phospholipids, the Rcs phosphorelay signal transduction system is activated, causing thermosensitive growth. The mutant grows poorly at 37 degrees C and lyses at 42 degrees C. We showed that the poor growth at 37 degrees C was corrected by disruption of the rcsA gene, which codes for a coregulator protein that interacts with the RcsB response regulator of the phosphorelay system. However, mutant cells still lysed when incubated at 42 degrees C even in the absence of RcsA. We conclude that the activated Rcs phosphorelay in the pgsA null mutant has both RcsA-dependent and -independent growth inhibitory effects. Since the Rcs system has been shown to positively regulate the essential cell division genes ftsA and ftsZ independently of RcsA, we measured cellular levels of the FtsZ protein, but found that the growth defect of the mutant at 42 degrees C did not involve a change in the level of this protein.     

Envelope disorder of Escherichia coli cells lacking phosphatidylglycerol

Phosphatidylglycerol, the most abundant acidic phospholipid in Escherichia coli, is considered to play specific roles in various cellular processes that are essential for cell viability. A null mutation of pgsA, which encodes phosphatidylglycerophosphate synthase, does indeed confer lethality. However, pgsA null mutants are viable if they lack the major outer membrane lipoprotein (Lpp) (lpp mutant) (S. Kikuchi, I. Shibuya, and K. Matsumoto, J. Bacteriol. 182:371-376, 2000). Here we show that Lpp expressed from a plasmid causes cell lysis in a pgsA lpp double mutant. The envelopes of cells harvested just before lysis could not be separated into outer and inner membrane fractions by sucrose density gradient centrifugation. In contrast, expression of a mutant Lpp (LppdeltaK) lacking the COOH-terminal lysine residue (required for covalent linking to peptidoglycan) did not cause lysis and allowed for the clear separation of the outer and inner membranes. We propose that in pgsA mutants LppdeltaK could not be modified by the addition of a diacylglyceryl moiety normally provided by phosphatidylglycerol and that this defect caused unmodified LppdeltaK to accumulate in the inner membrane. Although LppdeltaK accumulation did not lead to lysis, the accumulation of unmodified wild-type Lpp apparently led to the covalent linking to peptidoglycan, causing the inner membrane to be anomalously anchored to peptidoglycan and eventually leading to lysis. We suggest that this anomalous anchoring largely explains a major portion of the nonviable phenotypes of pgsA null mutants.     

Deletion of internal twenty-one amino acid residues of Escherichia coli prolipoprotein does not affect the formation of the murein-bound lipoprotein.

Mutation pgsA affecting the phosphatidylglycerol phosphate synthesis is lethal for all but certain E. coli strains such as strains deleted for the lpp gene or strains containing unmodifiable prolipoprotein like lppD14. Strain SD312 pgsA3 is tolerant to pgsA mutation, which suggests the lpp alleles in strain SD312 pgsA3 and its parental strain SD12 may be defective. DNA sequence analysis of the lpp genes in Escherichia coli strains SD12 and SD312 pgsA using asymmetric polymerase chain reaction showed that the lpp alleles in these two strains contained a 63 base pair deletion corresponding to the 37th to 57th codons of the wild-type lpp gene. [3H]Palmitate labeling of strains SD12 and SDS312 showed that the mutant lipoprotein in SD12 strain was modified with lipid, while the prolipoprotein in SD312 was not modified. The shortened mature lipoprotein in SD12 and the lipid-modified prolipoprotein in globomycin-treated SD12 were found to be covalently attached to the peptidoglycan, while the unmodified prolipoprotein in SD312 did not form significant amounts of murein-bound lipoprotein.     

Construction of a lethal mutation in the synthesis of the major acidic phospholipids of Escherichia coli

In order to determine if the major acidic phospholipids of Escherichia coli are essential to the organism, we constructed a null allele (pgsA30) of the pgsA gene thus rendering the organism incapable of synthesizing phosphatidylglycerol or cardiolipin. In strains carrying the pgsA30 allele cell viability, synthesis of gene product and the ability to synthesize the two major acidic phospholipids were dependent on the presence of a functional copy of the pgsA gene carried on a plasmid which was temperature-sensitive for replication. Growth ceased at the temperature restrictive for plasmid replication when the acidic phospholipid content dropped to about 10% of wild type levels which is slightly higher than the level reported in cells carrying the pgsA3 allele in a genetic background derived from strain SD12; the latter cells, which are capable of synthesizing low levels of acidic phospholipids, were previously shown to have no abnormal growth phenotype (Miyazaki, C., Kuroda, M., Ohta, A., and Shibuya, I. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7530-7534). The pgsA30 allele, unlike the pgsA3 allele, could not support growth in strain SD12. Neither allele could support growth in two other independently derived strains of E. coli. Therefore, there is a direct dependence of cell viability on a functional pgsA gene product. Strain SD12 appears to contain a suppressor which allows cells with a reduced capability to synthesize acidic phospholipid (pgsA3 allele) to grow, but cannot support growth in cells with a complete lack of synthetic capability (pgsA30 allele).     

Regulation of capsule synthesis and cell motility in Salmonella enterica by the essential gene igaA

Mutants of Salmonella enterica carrying the igaA1 allele, selected as able to overgrow within fibroblast cells in culture, are mucoid and show reduced motility. Mucoidy is caused by derepression of wca genes (necessary for capsule synthesis); these genes are regulated by the RcsC/YojN/RcsB phosphorelay system and by the RcsA coregulator. The induction of wca expression in an igaA1 mutant is suppressed by mutations in rcsA and rcsC. Reduced motility is caused by lowered expression of the flagellar master operon, flhDC, and is suppressed by mutations in rcsB or rcsC, suggesting that mutations in the igaA gene reduce motility by activating the RcsB/C system. A null igaA allele can be maintained only in an igaA(+)/igaA merodiploid, indicating that igaA is an essential gene. Lethality is suppressed by mutations in rcsB, rcsC, and yojN, but not in rcsA, suggesting that the viability defect of an igaA null mutant is mediated by the RcsB/RcsC system, independently of RcsA (and therefore of the wca genes). Because all the defects associated with igaA mutations are suppressed by mutations that block the RcsB/RcsC system, we propose a functional interaction between the igaA gene product and either the Rcs regulatory network or one of its regulated products.     

The Rcs phosphorelay is a cell envelope stress response activated by peptidoglycan stress and contributes to intrinsic antibiotic resistance

Gram-negative bacteria possess stress responses to maintain the integrity of the cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth, new peptidoglycan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identify Escherichia coli stress responses activated following inhibition of specific PBPs by the β-lactam antibiotics amdinocillin (mecillinam) and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance of E. coli to β-lactam antibiotics.     

Involvement of the ribulosephosphate epimerase gene in the dnaA and dnaR functions for initiation of chromosome replication in Escherichia coli

Initiation of replication of the Escherichia coli chromosome is rendered temperature-sensitive by the dnaR130 mutation in the prs gene that encodes phosphoribosylpyrophosphate synthetase. The thermosensitivity of the dnaR mutant is suppressed by a spontaneous mutation in rpe , the gene encoding ribulosephosphate epimerase. Disruption of the rpe gene reverses the thermosensitive growth of the dnaR mutant. The rpe -disrupted dnaR mutant exhibits extensive DNA synthesis at low and high temperatures, as does the dnaR  ⁺ rpe disruptant and the dnaR  ⁺ rpe ⁺ strain. The thermoresistant DNA synthesis in the rpe dnaR double mutant is dnaA dependent, because the dnaA167 mutation renders the synthesis thermosensitive. The rpe -knockout mutation abolishes the ability of the dnaA115 allele to complement the defect of the dnaA167 mutant with or without the dnaR mutation and diminishes the dnaR -complementing ability of the dnaR224 allele. These results show that the rpe product is involved in the functions of the products of both dnaA and dnaR for initiation of replication of the bacterial chromosome.     

Acetyl phosphate-sensitive regulation of flagellar biogenesis and capsular biosynthesis depends on the Rcs phosphorelay

As part of our attempt to map the impact of acetyl phosphate (acetyl approximately P) on the entire network of two-component signal transduction pathways in Escherichia coli, we asked whether the influence of acetyl approximately P on capsular biosynthesis and flagellar biogenesis depends on the Rcs phosphorelay. To do so, we performed a series of epistasis experiments: mutations in the components of the pathway that controls acetyl approximately P levels were combined with mutations in components of the Rcs phosphorelay. Cells that did not synthesize acetyl approximately P produced no capsule under normally permissive conditions, while those that accumulated acetyl approximately P synthesized capsule under conditions previously considered to be non-permissive. Acetyl approximately P-dependent capsular biosynthesis required both RcsB and RcsA, while the lack of RcsC restored capsular biosynthesis to acetyl approximately P-deficient cells. Similarly, acetyl approximately P-sensitive repression of flagellar biogenesis was suppressed by the loss of RcsB (but not of RcsA), while it was enhanced by the lack of RcsC. Taken together, these results show that both acetyl approximately P-sensitive activation of capsular biosynthesis and acetyl approximately P-sensitive repression of flagellar biogenesis require the Rcs phosphorelay. Moreover, they provide strong genetic support for the hypothesis that RcsC can function as either a kinase or a phosphatase dependent on environmental conditions. Finally, we learned that RcsB and RcsC inversely regulated the timing of flagellar biogenesis: rcsB mutants elaborated flagella prematurely, while rcsC mutants delayed their display of flagella. Temporal control of flagella biogenesis implicates the Rcs phosphorelay (and, by extension, acetyl approximately P) in the transition of motile, planktonic individuals into sessile biofilm communities.     

The RcsCDB signaling system and swarming motility in Salmonella enterica serovar typhimurium: dual regulation of flagellar and SPI-2 virulence genes

The Rcs phosphorelay is a multicomponent signaling system that positively regulates colanic acid synthesis and negatively regulates motility and virulence. We have exploited a spontaneously isolated mutant, IgaA(T191P), that is nearly maximally activated for the Rcs system to identify a vast set of genes that respond to the stimulation, and we report new regulatory properties of this signaling system in Salmonella enterica serovar Typhimurium. Microarray data show that the Rcs system normally functions as a positive regulator of SPI-2 and other genes important for the growth of Salmonella in macrophages, although when highly activated the system completely represses the SPI-1/SPI-2 virulence, flagellar, and fimbrial biogenesis pathways. The auxiliary protein RcsA, which works with RcsB to positively regulate colanic acid and other target genes, not only stimulates but also antagonizes the positive regulation of many genes in the igaA mutant. We show that RcsB represses motility through the RcsB box in the promoter region of the master operon flhDC and that RcsA is not required for this regulation. Curiously, RcsB selectively stimulates expression of the flagellar type 3 secretion genes fliPQR; an RcsAB box located downstream of fliR influences this regulation. We show that excess colanic acid impairs swimming and inhibits swarming motility, consistent with the inverse regulation of the two pathways by the Rcs system.     

Rcs双组分调节系统对细菌环境应答的分子调控研究进展

荚膜异多糖酸合成调节(Regulator of Capsule Synthesis,Rcs)系统是存在于许多肠杆菌科细菌中非典型的双组分调节系统,由3种核心蛋白(跨膜感应激酶RcsC、跨膜蛋白RcsD和响应调节剂RcsB)及多种辅助蛋白共同构成。Rcs系统能整合环境信号、调节基因表达并改变细菌的生理行为。近年来,对细菌Rcs系统环境应答机制的探索成为一个新的研究热点。本文重点综述Rcs系统上游信号的感知与传导、Rcs系统调节的下游靶基因及其生命现象,以期能增进对细菌Rcs系统的认识,同时为细菌的安全控制、感染预防和治疗新方案的开发提供参考。     

Chromosome-plasmid interaction in Escherichia coli K-12 carrying a thermosensitive plasmid, Rts1, in autonomous and in integrated states.

An Hfr strain of Escherichia coli K-12 was obtained by integrative suppression with a thermosensitive plasmid, Rts1. The R plasmid was integrated into the chromosome between rif and thr, and transfer of the chromosome occurred counterclockwise. The thermosensitivity of host cell growth due to the dnaA mutation was markedly but not completely reduced in this integratively suppressed Hfr strain. When the dnaA mutation was removed by transducing the dnaA+ genome to this Hfr, the thermosensitivity of cell growth due to existence of Rts1 was suppressed in contrast to strains carrying it autonomously. Thermosensitivity of cell growth appeared again when the plasmid was detached from the chromosome to exist autonomously. Contrary to the effect on cell growth, the transfer of the chromosome and the plasmid itself and the ability to "restrict" T-even phages were still thermosensitive in all of these strains carrying Rts1, irrespective of its state of existence. The detached plasmid as well as the original Rts1 were segregated upon growth at 42 C. These data are discussed in relation to chromosome-plasmid interaction. One of the most important conculusions is that some plasmid genes, related to their replication, are phenotypically suppressed by the chromosome when it is integrated.     

Alteration of the phospholipid composition of Escherichia coli through genetic manipulation

In order to study the function of individual phospholipids, we have constructed a strain of Escherichia coli in which the ratio of phosphatidylethanolamine to phosphatidylglycerol plus cardiolipin can be regulated. In this strain (HDL1001) the normal expression of the phosphatidylglycerophosphate synthase does not occur due to the presence of the pgsA30 allele (Heacock, P. N., and Dowhan, W. (1987) J. Biol. Chem. 262, 13044-13049). A second chromosomal copy of the pgsA gene is fused to the lacOP region in single copy within the lac operon. Strain HDL1001 is absolutely dependent for growth on an inducer of the lac operon. In addition, the level of the pgsA gene product, the content of the two major acidic phospholipids, and the growth rate are dependent on the level of inducer in the growth medium. Cells remain viable in the absence of inducer as evidenced by a rapid return to normal growth after the readdition of inducer. The growth rate and phospholipid composition are affected only after the level of phosphatidylglycerophosphate synthase drops below about 15% of normal levels; both phosphatidic acid and (d)CDP-diacylglycerol also begin to increase to significant levels. At the point of cell arrest the level of the major acidic phospholipids is reduced by about 90% of wild type levels.     

DjlA negatively regulates the Rcs signal transduction system in Escherichia coli

The Rcs signal transduction system of Escherichia coli regulating capsular polysaccharide synthesis (cps) genes is activated by overexpression of the djlA gene encoding a cytoplasmic membrane-anchored DnaJ-like protein. However, by monitoring the expression of a cpsB'-lac fusion in pgsA- and mdoH-null mutants in which the Rcs system is activated, we found that the Rcs activity was further increased by deletion of djlA and decreased by low-level extrachromosomal expression of djlA. Furthermore, deletion of djlA in a wild-type strain led to small but significant increase of the basal-level activity of the Rcs system. These results demonstrate that DjlA functions as a negative regulator of the Rcs system unless abnormally overproduced.