Activation of the Rcs signal transduction system is responsible for the thermosensitive growth defect of an Escherichia coli mutant lacking phosphatidylglycerol and cardiolipin

The lethal effect of an Escherichia coli pgsA null mutation, which causes a complete lack of the major acidic phospholipids, phosphatidylglycerol and cardiolipin, is alleviated by a lack of the major outer membrane lipoprotein encoded by the lpp gene, but an lpp pgsA strain shows a thermosensitive growth defect. Using transposon mutagenesis, we found that this thermosensitivity was suppressed by disruption of the rcsC, rcsF, and yojN genes, which code for a sensor kinase, accessory positive factor, and phosphotransmitter, respectively, of the Rcs phosphorelay signal transduction system initially identified as regulating the capsular polysaccharide synthesis (cps) genes. Disruption of the rcsB gene coding for the response regulator of the system also suppressed the thermosensitivity, whereas disruption of cpsE did not. By monitoring the expression of a cpsB'-lac fusion, we showed that the Rcs system is activated in the pgsA mutant and is reverted to a wild-type level by the rcs mutations. These results indicate that envelope stress due to an acidic phospholipid deficiency activates the Rcs phosphorelay system and thereby causes the thermosensitive growth defect independent of the activation of capsule synthesis.     

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.     

Viability of an Escherichia coli pgsA null mutant lacking detectable phosphatidylglycerol and cardiolipin

Phosphatidylglycerol, the most abundant acidic phospholipid in Escherichia coli, has been considered to play specific roles in various cellular processes and is believed to be essential for cell viability. It is functionally replaced in some cases by cardiolipin, another abundant acidic phospholipid derived from phosphatidylglycerol. However, we now show that a null pgsA mutant is viable, if the major outer membrane lipoprotein is deficient. The pgsA gene normally encodes phosphatidylglycerophosphate synthase that catalyzes the committed step in the biosynthesis of these acidic phospholipids. In the mutant, the activity of this enzyme and both phosphatidylglycerol and cardiolipin were not detected (less than 0.01% of total phospholipid, both below the detection limit), although phosphatidic acid, an acidic biosynthetic precursor, accumulated (4.0%). Nonetheless, the null mutant grew almost normally in rich media. In low-osmolarity media and minimal media, however, it could not grow. It did not grow at temperatures over 40 degrees C, explaining the previous inability to construct a null pgsA mutant (W. Xia and W. Dowhan, Proc. Natl. Acad. Sci. USA 92:783-787, 1995). Phosphatidylglycerol and cardiolipin are therefore nonessential for cell viability or basic life functions. This notion allows us to formulate a working model that defines the physiological functions of acidic phospholipids in E. coli and explains the suppressing effect of lipoprotein deficiency.     

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).     

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.     

Dispensable nature of phosphatidylglycerol in Escherichia coli: dual roles of anionic phospholipids

The major anionic phospholipids of Escherichia coli, phosphatidylglycerol (PG) and cardiolipin (CL), have been considered to be indispensable for essential cellular functions, such as the initiation of DNA replication and translocation of proteins across the cytoplasmic membrane. However, we successfully constructed a null pgsA mutant of E. coli that had undetectable levels of PG and CL if the major outer membrane lipoprotein was deficient, clearly indicating that these anionic phospholipids are not indispensable. In the null mutant, we observed the accumulation of phosphatidic acid, an acidic biosynthetic precursor. This suggests a functionally substitutable nature of these anionic phospholipids and allows us to formulate a dual role model for the physiological roles of the anionic phospholipids in E. coli. The anionic phospholipids may play dual roles in E. coli as (i) substrates for head group-specific enzyme reactions, albeit the viability of null PG mutants indicates that the products of head group-specific reactions are not essential; and (ii) those that are replaceable, partly or entirely, by other phospholipids bearing net negative charges, because of their rather loose head group specificity. These two aspects of the physiological roles of anionic phospholipids are discussed with special reference to the phospholipids of other bacteria and eukaryotic organelles.     

Increases in acidic phospholipid contents specifically restore protein translocation in a cold-sensitive secA or secG null mutant.

Both the secAcsR11 and DeltasecG::kan mutations cause cold-sensitive growth, although the growth defect due to the latter mutation occurs in a strain-specific manner. Overexpression of pgsA encoding phosphatidylglycerophosphate synthase suppresses the growth defects of the two mutants. We investigated the mechanism underlying the pgsA-dependent suppression of the two mutations using purified mutant SecA and inverted membrane vesicles (IMVs) prepared from pgsA-overexpressing cells. The acidic phospholipid content increased by about 10% upon pgsA overexpression. This increase resulted in the stimulation of proOmpA translocation only when mutant SecA or SecG-depleted IMVs were used. The translocation-coupled ATPase activity of SecA was significantly defective with the mutant SecA or SecG-depleted IMVs, but it recovered to a near normal level when the acidic phospholipid level was increased. The stimulation of ATPase activity was observed only at low temperature. The steady-state level of membrane-inserted SecA was low with the mutant SecA or SecG-depleted IMVs, and it decreased further upon the increase in the acidic phospholipid content. However, the level of SecA insertion markedly increased upon the inhibition of SecA deinsertion by the addition of beta,gamma-imido adenosine 5'-triphosphate (AMP-PNP), especially with IMVs containing increased levels of acidic phospholipids. These results indicate that the increase in the level of acidic phospholipids stimulates the SecA cycle in the two mutants by facilitating both the insertion and deinsertion of SecA.     

Phosphatidylinositol is an essential phospholipid of mycobacteria

Phosphatidylinositol (PI) and metabolically derived products such as the phosphatidylinositol mannosides and linear and mature branched lipomannan and lipoarabinomannan are prominent phospholipids/lipoglycans of Mycobacterium sp. believed to play important roles in the structure and physiology of the bacterium as well as during host infection. To determine if PI is an essential phospholipid of mycobacteria, we identified the pgsA gene of Mycobacterium tuberculosis encoding the phosphatidylinositol synthase enzyme and constructed a pgsA conditional mutant of Mycobacterium smegmatis. The ability of this mutant to synthesize phosphatidylinositol synthase and subsequently PI was dependent on the presence of a functional copy of the pgsA gene carried on a thermosensitive plasmid. The mutant grew like the control strain under permissive conditions (30 degrees C), but ceased growing when placed at 42 degrees C, a temperature at which the rescue plasmid is lost. Loss of cell viability at 42 degrees C was observed when PI and phosphatidylinositol dimannoside contents dropped to approximately 30 and 50% of the wild-type levels, respectively. This work provides the first evidence of the essentiality of PI to the survival of mycobacteria. PI synthase is thus an essential enzyme of Mycobacterium that shows promise as a drug target for anti-tuberculosis therapy.     

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.     

Isolation and expression of the Rhodobacter sphaeroides gene (pgsA) encoding phosphatidylglycerophosphate synthase.

The Rhodobacter sphaeroides pgsA gene (pgsARs), encoding phosphatidylglycerophosphate synthase (PgsARs), was cloned, sequenced, and expressed in both R. sphaeroides and Escherichia coli. As in E. coli, pgsARs is located immediately downstream of the uvrC gene. Comparison of the deduced amino acid sequences revealed 41% identity and 69% similarity to the pgsA gene of E. coli, with similar homology to the products of the putative pgsA genes of several other bacteria. Comparison of the amino acid sequences of a number of enzymes involved in CDP-diacylglycerol-dependent phosphatidyltransfer identified a highly conserved region also found in PgsARs. The pgsARs gene carried on multicopy plasmids was expressed in R. sphaeroides under the direction of its own promoter, the R. sphaeroides rrnB promoter, and the E. coli lac promoter, and this resulted in significant overproduction of PgsARs activity. Expression of PgsARs activity in E. coli occurred only with the E. coli lac promoter. PgsARs could functionally replace the E. coli enzyme in both a point mutant and a null mutant of E. coli pgsA. Overexpression of PgsARs in either E. coli or R. sphaeroides did not have dramatic effects on the phospholipid composition of the cells, suggesting regulation of the activity of this enzyme in both organisms.     

Genetic evidence for functional interaction of the Escherichia coli signal recognition particle receptor with acidic lipids in vivo

The mechanism underlying the interaction of the Escherichia coli signal recognition particle receptor FtsY with the cytoplasmic membrane has been studied in detail. Recently, we proposed that FtsY requires functional interaction with inner membrane lipids at a late stage of the signal recognition particle pathway. In addition, an essential lipid-binding α-helix was identified in FtsY of various origins. Theoretical considerations and in vitro studies have suggested that it interacts with acidic lipids, but this notion is not yet fully supported by in vivo experimental evidence. Here, we present an unbiased genetic clue, obtained by serendipity, supporting the involvement of acidic lipids. Utilizing a dominant negative mutant of FtsY (termed NG), which is defective in its functional interaction with lipids, we screened for E. coli genes that suppress the negative dominant phenotype. In addition to several unrelated phenotype-suppressor genes, we identified pgsA, which encodes the enzyme phosphatidylglycerophosphate synthase (PgsA). PgsA is an integral membrane protein that catalyzes the committed step to acidic phospholipid synthesis, and we show that its overexpression increases the contents of cardiolipin and phosphatidylglycerol. Remarkably, expression of PgsA also stabilizes NG and restores its biological function. Collectively, our results strongly support the notion that FtsY functionally interacts with acidic lipids.     

Characterization of Null Mutants of the RAD55 Gene of Saccharomyces cerevisiae: Effects of Temperature, Osmotic Strength and Mating Type

RAD55 belongs to a group of genes required for resistance to ionizing radiation, RAD50-RAD57, which are thought to define a pathway of recombinational repair. Since all four alleles of RAD55 are temperature conditional (cold sensitive) for their radiation phenotype, we investigated the phenotype produced by null mutations in the RAD55 gene, constructed in vitro and transplaced to the yeast chromosome. The X-ray sensitivity of these null mutant strains was surprisingly suppressed by increased temperature, osmotic strength of the growth medium and heterozygosity at the mating-type locus. These first two properties, temperature conditionality and osmotic remediability, are commonly associated with missense mutations; these rad55 null mutants are unique in that they exhibit these properties although the mutant gene cannot be expressed. X-ray-induced mitotic recombination was also cold sensitive in rad55 mutant diploids. Although mitotic growth was unaffected in these strains, meiosis was a lethal event at both high and low temperatures. Whereas the phenotype of rad55 null mutants is consistent with a role of RAD55 in recombination and recombinational repair, there is evidence for considerable RAD55-independent recombination, at least in mitotic cells, which is influenced by temperature and MAT. We discuss models for the role of RAD55 in recombination to explain the unusual properties of rad55 mutants.     

Mechanism of phage-induced lysis in pneumococci

Earlier studies have suggested the possible role of host autolytic enzyme in the release of progeny phage from Dp-1 infected pneumococci. Several new experiments described here reinforce this notion. Specifically, the resistance of an autolysis-defective mutant to infection at low phage to cell ratios could be eliminated by prior 'coating' of the host bacteria with pneumococcal autolysin isolated from wild-type cells. Similar, productive infection was also possible by lowering the temperature of incubation to 30 degrees C, a condition that leads to a partial activation of the thermosensitive residual autolysin in the mutant cells. Other experiments, however, clearly indicate the role of the newly discovered phage-associated lysin (PAL), reported in the accompanying communication, in bacteriophage release and culture lysis; specifically, lysis was stimulated by reducing agents and inhibited by cardiolipin. It seems that both the host-related and the PAL activities are involved with Dp-1 induced lysis of pneumococci.     

Alteration in the contents of unsaturated fatty acids in dnaA mutants of Escherichia coli

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli , has a high affinity for acidic phospholipids containing unsaturated fatty acids. We have examined here the fatty acid composition of phospholipids in dnaA mutants. A temperature-sensitive dnaA46 mutant showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) at 42°C (non-permissive temperature) and at 37°C (semi-permissive temperature), but not at 28°C (permissive temperature), compared with the wild-type strain. Plasmid complementation analysis revealed that the dnaA46 mutation is responsible for the phenotype. Other temperature-sensitive dnaA mutants showed similar results. On the other hand, a cold-sensitive dnaAcos mutant, in which overinitiation of DNA replication occurs at low temperature (28°C), showed a higher level of unsaturation of fatty acids at 28°C. Based on these observations, we discuss the role of phospholipids in the regulation of the activity of DnaA protein.     

A single amino acid substitution in the phosphoprotein of respiratory syncytial virus confers thermosensitivity in a reconstituted RNA polymerase system

The single amino acid change Gly172 to Ser in the phosphoprotein (P) of respiratory syncytial virus (RSV) has previously been shown to be responsible for the thermosensitivity and protein-negative phenotype of tsN19, a mutant of the B subgroup RSN-2 strain. This single change was inserted into the P gene of the A subgroup virus RSS-2, and the resulting phenotype was observed in a plasmid-driven reconstituted RSV RNA polymerase system. Expression from a genome analogue containing two reporter genes was thermosensitive when directed by plasmids containing the N, L, M2, and mutant P genes cloned under the control of T7 promoters. Analysis of RNA synthesis showed that mutant P protein was unable to produce genome, antigenome, or mRNA at the restrictive temperature. At a semipermissive temperature, genome, antigenome, and mRNA synthesis were all reduced, 6- to 30-fold, relative to synthesis directed by a wild-type P plasmid. Binding of the mutant P protein to N protein in the absence of other viral proteins was unaffected by temperature, indicating that the lesion did not produce a large enough structural change to disrupt this binding. These data suggest that the plasmid rescue system is suitable for investigation of the role of thermosensitive mutations in RSV polymerase components in RNA synthesis.     

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 system is essential for pathogenicity in Erwinia amylovora

The Rcs phosphorelay system is a modified two-component signal transduction system found exclusively in Enterobacteriaceae . In this study, we characterized the roles of the Rcs system in Erwinia amylovora , a highly virulent and necrogenic enterobacterium causing fire blight disease on rosaceous plants. Our results showed that rcsB , rcsC , rcsD and rcsBD mutants were non-pathogenic on immature pear fruit. The bacterial growth of these mutants was also greatly reduced compared with that of the wild-type strain in immature pear fruit. In an in vitro amylovoran assay, rcsB and rcsD mutants were deficient in amylovoran production, whereas the rcsC mutant exhibited higher amylovoran production than that of the wild-type. Consistent with amylovoran production, expression of the amylovoran biosynthetic gene amsG , using green fluorescent protein as a reporter, was not detectable in rcsB , rcsD and rcsBD mutants both in vitro and in vivo . The expression of amsG in vitro was higher in the rcsC mutant than in the wild-type, whereas its expression in vivo was higher in the wild-type than in the rcsC mutant. In addition, rcs mutants were more susceptible to polymyxin B treatment than the wild-type, suggesting that the Rcs system conferred some level of resistance to polymyxin B. Furthermore, rcs mutants showed irregular and slightly reduced motility on swarming plates. Together, these results indicate that the Rcs system plays a major role in virulence and survival of E. amylovora in immature pear fruit.