Stabilization of cell wall proteins in Bacillus subtilis: a proteomic approach

Even though cell wall proteins of Bacillus subtilis are characterized by specific cell wall retention signals, some of these are also components of the extracellular proteome. In contrast to the majority of extracellular proteins, wall binding proteins disappeared from the extracellular proteome during the stationary phase and are subjected to proteolysis. Thus, the extracellular proteome of the multiple protease-deficient strain WB700 was analyzed which showed an increased stability of secreted WapA processing products during the stationary phase. In addition, stabilization of the WapA processing products was observed also in a sigD mutant strain which is impaired in motility and cell wall turnover. Next, we analyzed if proteins that can be extracted from B. subtilis cell walls are stabilized in the WB700 strain as well as in the sigD mutant. Thus, the cell wall proteome of B. subtilis wild type was defined showing most abundantly cell wall binding proteins (CWBPs) resulting from the WapA and WprA precursor processing. The inactivation of extracellular proteases as well as SigmaD caused an increase of CWBP105 and a decrease of CWBP62 in the cell wall proteome. We conclude that WapA processing products are substrates for the extracellular proteases which are stabilized in the absence of sigD due to an impaired cell wall turnover.     

Simultaneous inactivation of the wprA and dltB genes of Bacillus subtilis reduces the yield of alpha-amylase

AIMS: In Gram-positive bacteria, signal peptide-bearing secretory proteins are translocated through the cytoplasmic membrane and fold into their native conformation on the outside of the cell. The products of the Bacillus subtilis wprA and dltB genes separately influence post-translocational stages of the secretion process by mediating proteolytic degradation and folding of secretory proteins. Inactivation of either wprA or dltB in B. subtilis increases the yield of secretory proteins released into the culture medium in an intact and biologically active conformation. The aim of this work was to study the combined influence of these genes. METHODS AND RESULTS: A wprA/dltB double mutant was constructed, but did not have an additive effect on secretion and caused a significant reduction in the yield of alpha-amylase. CONCLUSIONS AND SIGNIFICANCE: The activities of the wprA gene and the dlt operon interact in a negative way to influence the growth cycle and protein secretion. The mechanism by which this may occur, and its potential significance for the secretion of native and non-native proteins from B. subtilis and related bacteria, is discussed.     

Enhancement of secretion and extracellular stability of staphylokinase in Bacillus subtilis by wprA gene disruption

Staphylokinase (SAK), a polypeptide secreted by Staphylococcus aureus, is a plasminogen activator with a therapeutic potential in thrombosis diseases. A Bacillus subtilis strain which is multiply deficient in exoproteases was transformed by an expression plasmid carrying a promoter and a signal sequence of subtilisin fused in frame with the sak open reading frame. However, the amount of SAK secretion was marginal (45 mg/liter). In contrast, disruption of the wprA gene, which encodes a subtilisin-type protease, strongly promoted the production of SAK in the stationary phase (181 mg/liter). In addition, the extracellular stability of mature SAK was dramatically enhanced. These data indicate a significant role of the wprA gene product in degrading foreign proteins, both during secretion and in the extracellular milieu.     

Improved expression of human interleukin-2 in high-cell-density fermentor cultures of Escherichia coli K-12 by a phosphotransacetylase mutant

A fluoroacetate-resistant mutant of Escherichia coli K-12 (MM-294) accumulated less acetate in the medium during growth to high cell density in fermentor cultures and was shown to be defective in its phosphotransacetylase activity. The mutant had an improved ability to continue growing during induction of interleukin-2 (IL-2) synthesis, and in fermentor cultures it gave a higher level of specific IL-2 accumulation than its parent during expression under control of the temperature-sensitive pL promoter. In flask cultures at lower cell density, the mutant again produced less acetate than the parent, although both showed a much lower level of acetate accumulation than that seen in fermentors at high cell density. Both showed a higher specific expression level of IL-2 in flask cultures, and there was a greater difference between the mutant and its parent in the final extent of specific IL-2 accumulation in fermentor cultures compared with flask cultures. Thus, the concentration of acetate in the medium, which was much higher in fermentor cultures (greater than or equal to 300 mM after 5 h of induction) than in flask cultures (less than or equal to mM) of the parent organism, was a significant factor in limiting expression of the heterologous protein product, IL-2. The acetate kinase-phosphotransacetylase pathway was therefore a major source of acetate formation in these cultures. Blocking this pathway improved accumulation of IL-2 and did not slow growth.     

Improved expression of human interleukin-2 in high-cell-density fermentor cultures of Escherichia coli K-12 by a phosphotransacetylase mutant.

A fluoroacetate-resistant mutant of Escherichia coli K-12 (MM-294) accumulated less acetate in the medium during growth to high cell density in fermentor cultures and was shown to be defective in its phosphotransacetylase activity. The mutant had an improved ability to continue growing during induction of interleukin-2 (IL-2) synthesis, and in fermentor cultures it gave a higher level of specific IL-2 accumulation than its parent during expression under control of the temperature-sensitive pL promoter. In flask cultures at lower cell density, the mutant again produced less acetate than the parent, although both showed a much lower level of acetate accumulation than that seen in fermentors at high cell density. Both showed a higher specific expression level of IL-2 in flask cultures, and there was a greater difference between the mutant and its parent in the final extent of specific IL-2 accumulation in fermentor cultures compared with flask cultures. Thus, the concentration of acetate in the medium, which was much higher in fermentor cultures (greater than or equal to 300 mM after 5 h of induction) than in flask cultures (less than or equal to mM) of the parent organism, was a significant factor in limiting expression of the heterologous protein product, IL-2. The acetate kinase-phosphotransacetylase pathway was therefore a major source of acetate formation in these cultures. Blocking this pathway improved accumulation of IL-2 and did not slow growth.     

The alternative sigma factor, sigmaS, affects polyhydroxyalkanoate metabolism in Pseudomonas putida

To determine whether the stationary sigma factor, sigma(S), influences polyhydroxyalkanoate metabolism in Pseudomonas putida KT2440, an rpoS-negative mutant was constructed to evaluate polyhydroxyalkanoate accumulation and expression of a translational fusion to the promoter region of the genes that code for polyhydroxyalkanoate synthase 1 (phaC1) and polyhydroxyalkanoate depolymerase (phaZ). By comparison with the wild-type, the rpoS mutant showed a higher polyhydroxyalkanoate degradation rate and increased expression of the translational fusion during the stationary growth phase. These results suggest that sigma(S) might control the genes involved in polyhydroxyalkanoate metabolism, possibly in an indirect manner. In addition, survival and oxidative stress assays performed under polyhydroxyalkanoate- and nonpolyhydroxyalkanoate- accumulating conditions demonstrated that the accumulated polyhydroxyalkanoate increased the survival and stress tolerance of the rpoS mutant. According to this, polyhydroxyalkanoate accumulation would help cells to overcome the adverse conditions encountered during the stationary phase in the strain that lacks RpoS.     

A defect of the vacuolar putative lipase Atg15 accelerates degradation of lipid droplets through lipolysis

Lipid droplets (LDs) are the conserved organelles for the deposit of neutral lipids, and function as reservoirs of membrane and energy sources. To date, functional links between autophagy and LD dynamics have not been fully elucidated. Here, we report that a vacuolar putative lipase, Atg15, required for degradation of autophagic bodies, is crucial for the maintenance of LD amount in the yeast Saccharomyces cerevisiae in the stationary phase. Mutant analyses revealed that the putative lipase motif and vacuolar localization of Atg15 are important for the maintenance of LD amount. Loss of autophagosome formation by simultaneous deletion of core ATG genes cancelled the reduction in the LD amount in ATG15-deleted cells, indicating that degradation of autophagic bodies accounts for the functional involvement of Atg15 in LD dynamics. The reduced level of LDs in the mutant strain was dependent on Tgl3 and Tgl4, major lipases for lipolysis in S. cerevisiae. An altered phosphorylation status of Tgl3, higher accumulation of Tgl4, and closer associations of Tgl3 and Tgl4 with LDs were detected in the ATG15-deleted cells. Furthermore, increased levels of downstream metabolites of lipolysis in the mutant strain strongly suggested enhanced lipolytic activity caused by loss of ATG15. Our data provide evidence for a novel link between autophagic flux and LD dynamics integrated with Atg15 activity.     

The alternative sigma factor, σS, affects polyhydroxyalkanoate metabolism in Pseudomonas putida

To determine whether the stationary sigma factor, σ^S, influences polyhydroxyalkanoate metabolism in Pseudomonas putida KT2440, an rpoS -negative mutant was constructed to evaluate polyhydroxyalkanoate accumulation and expression of a translational fusion to the promoter region of the genes that code for polyhydroxyalkanoate synthase 1 ( phaC1 ) and polyhydroxyalkanoate depolymerase ( phaZ ). By comparison with the wild-type, the rpoS mutant showed a higher polyhydroxyalkanoate degradation rate and increased expression of the translational fusion during the stationary growth phase. These results suggest that σ^S might control the genes involved in polyhydroxyalkanoate metabolism, possibly in an indirect manner. In addition, survival and oxidative stress assays performed under polyhydroxyalkanoate- and nonpolyhydroxyalkanoate- accumulating conditions demonstrated that the accumulated polyhydroxyalkanoate increased the survival and stress tolerance of the rpoS mutant. According to this, polyhydroxyalkanoate accumulation would help cells to overcome the adverse conditions encountered during the stationary phase in the strain that lacks RpoS.     

Metabolic differences betweenEscherichia coli cultures growing aerobically and anaerobically in the presence of fluoroacetate

The amount of citrate and pyruvate increased during the stationary phase of anEscherichia coli B culture growing in a synthetic medium, aerobically in the presence of glucose. In an anaerobic culture the amount of citrate was minute and did not rise during the stationary phase; the level of pyruvate in the stationary phase rose only slightly. Fluoroacetate blocked the growth of cells both aerobically and anaerobically. Cultures growing aerobically in the presence of fluoroaeetate displayed an increased accumulation of citrate as compared with the control. In anaerobic cultures citrate did not accumulate in the presence of inhibitory concentrations of fluoroacetate. In the presence of 2mm fluoroacetate cells grew aerobically somewhat more slowly at first, then inhibition ceased and finally the growth yield was greater than in the control. The obtained data indicate that citrate accumulated at first was partly utilized for growth under these conditions. The results are discussed from the point of view of differences in the metabolism of aerobically and anaerobically grown cells.     

Synthesis citric and isocitric acids by Yarrowia lipolytica during yeasts growth on rapeseed oils

The native strain Yarrowia lipolytica VKM Y-2373 grown in a complete medium exhibited the maximum lipase activity at the concentration of rapeseed oil of at least 5.0 g/l. In the course of yeast growth, no considerable changes were observed in the glycerol concentration, the proportions of the major free fatty acids formed via oil hydrolysis, or the fatty acid composition of oil. Under nitrogen limitation of cell growth, the accumulation of citric acids reached 77.1 g/l with predominance of isocitric acid at pH 6.0, whereas at pH 4.5, almost equal amounts of citric and isocitric acids were produced. Cultivation of the mutant strain Y. lipolytica N 1 at pH 4.5 resulted in the predominant accumulation of citric acid (66.6 g/l) with an insignificant amount of isocitric acid. In the period of intense acid synthesis, high production of lipase was observed.     

Lipoprotein lipase expression level influences tissue clearance of chylomicron retinyl ester

Approximately 25% of postprandial retinoid is cleared from the circulation by extrahepatic tissues. Little is known about physiologic factors important to this uptake. We hypothesized that lipoprotein lipase (LpL) contributes to extrahepatic clearance of chylomicron vitamin A. To investigate this, [3H]retinyl ester-containing rat mesenteric chylomicrons were injected intravenously into induced mutant mice and nutritionally manipulated rats. The tissue sites of uptake of 3H label by wild type mice and LpL-null mice overexpressing human LpL in muscle indicate that LpL expression does influence accumulation of chylomicron retinoid. Skeletal muscle from mice overexpressing human LpL accumulated 1.7- to 2.4-fold more 3H label than wild type. Moreover, heart tissue from mice overexpresssing human LpL, but lacking mouse LpL, accumulated less than half of the 3H-label taken up by wild type heart. Fasting and heparin injection, two factors that increase LpL activity in skeletal muscle, increased uptake of chylomicron [3H] retinoid by rat skeletal muscle. Using [3H]retinyl palmitate and its non-hydrolyzable analog retinyl [14C]hexadecyl ether incorporated into Intralipid emulsions, the importance of retinyl ester hydrolysis in this process was assessed. We observed that 3H label was taken up to a greater extent than 14C label by rat skeletal muscle, suggesting that retinoid uptake requires hydrolysis.In summary, for each of our experiments, the level of lipoprotein lipase expression in skeletal muscle, heart, and/or adipose tissue influenced the amount of [3H]retinoid taken up from chylomicrons and/or their remnants.     

NS2 is required for efficient translation of viral mRNA in minute virus of mice-infected murine cells.

Detailed analysis of five NS2 mutants of the autonomous parvovirus minute virus of mice (MVMp) has revealed the following. At low multiplicities of infection, NS2 mutants killed NB324K cells as well as wild-type (wt) MVM did and grew to high titers, while in contrast they grew poorly and did not readily kill murine A9 cells. Following CaPO4 transfection of murine fibroblasts, NS2 mutant infectious clones generated approximately 10-fold less monomer replicative-form DNA than wt and no detectable progeny single-stranded DNA. On nonmurine semipermissive NB324K cells, however, these mutant plasmid clones generated near wt levels of all replicative DNA forms. After infection of highly synchronized murine fibroblasts by NS2 mutant virus at inputs equivalent to those of the wt, mutant monomer replicative-form DNA was decreased 5- to 10-fold compared with that of the wt, and progeny single-stranded DNA accumulation was decreased to an even greater extent. Both total and cytoplasmic NS2 mutant RNA was decreased, but the amount of total viral mRNA generated, relative to accumulated viral DNA in the same experiments, was similar to that seen in wt infection. The accumulation of virus-generated proteins was also decreased in NS2 mutant infection; however, the magnitude of this decrease, compared with that of wt infections, was significantly greater than the concomitant decrease in mutant-generated levels of accumulated cytoplasmic RNA, and this effect was most dramatic for VP2. There was no such disparity between the relative accumulation of mutant-generated RNA and protein in cells permissive for the growth of these mutants. These results suggest that translation of MVM viral RNA is specifically reduced in NS2 mutant infection of restrictive cells. Because the affected viral proteins are required for the efficient production of viral replicative DNA forms, these results reveal a fundamental, although perhaps not the only, role for NS2 in parvovirus infection.     

Identification and Inactivation of Three Group 2 Sigma Factor Genes in Anabaena sp. Strain PCC 7120

Three new Anabaena sp. strain PCC 7120 genes encoding group 2 alternative sigma factors have been cloned and characterized. Insertional inactivation of sigD, sigE, and sigF genes did not affect growth on nitrate under standard laboratory conditions but did transiently impair the abilities of sigD and sigE mutant strains to establish diazotrophic growth. A sigD sigE double mutant, though proficient in growth on nitrate and still able to differentiate into distinct proheterocysts, was unable to grow diazotrophically due to extensive fragmentation of filaments upon nitrogen deprivation. This double mutant could be complemented by wild-type copies of sigD or sigE, indicating some degree of functional redundancy that can partially mask phenotypes of single gene mutants. However, the sigE gene was required for lysogenic development of the temperate cyanophage A-4L. Several other combinations of double mutations, especially sigE sigF, caused a transient defect in establishing diazotrophic growth, manifested as a strong and prolonged bleaching response to nitrogen deprivation. We found no evidence for developmental regulation of the sigma factor genes. luxAB reporter fusions with sigD, sigE, and sigF all showed slightly reduced expression after induction of heterocyst development by nitrogen stepdown. Phylogenetic analysis of cyanobacterial group 2 sigma factor sequences revealed that they fall into several subgroups. Three morphologically and physiologically distant strains, Anabaena sp. strain PCC 7120, Synechococcus sp. strain PCC 7002, and Synechocystis sp. strain PCC 6803 each contain representatives of four subgroups. Unlike unicellular strains, Anabaena sp. strain PCC 7120 has three additional group 2 sigma factors that cluster in subgroup 2.5b, which is perhaps specific for filamentous or heterocystous cyanobacteria.     

5′-Nucleotidase Activity in Improved Inosine-producing Mutants of Bacillus subtilis

An inosine- and guanosine-producing strain, AJ11100, of Bacillus subtilis could not grow in the minimum medium supplemented with 50 µg of sulfaguanidine per ml. When sulfaguanidine resistant mutants were derived from AJ11100, the sulfaguanidine resistance was frequently accompanied by xanthine requirement. All the xanthine auxotrophic mutants required a large amount of xanthine for cell growth and inosine accumulation. Revertants were then derived from one of the xanthine auxotrophic mutants, AJ11101, and improved inosine producers were obtained. The best mutant, AJ11102, accumulated 20.6 g of inosine per liter.

Furthermore, enzyme activities of inosine 5′-monophosphate (IMP) dehydrogenase, 5′-nucleotidase and phosphoribosyl pyrophosphate (PRPP) amidotransferase were assayed to investigate why AJ11102 accumulated an increased amount of inosine. The results showed that the increase of specific activity of 5′-nucleotidase contributed much to the increased accumulation of inosine.     

FadD is required for utilization of endogenous fatty acids released from membrane lipids

FadD is an acyl coenzyme A (CoA) synthetase responsible for the activation of exogenous long-chain fatty acids (LCFA) into acyl-CoAs. Mutation of fadD in the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti promotes swarming motility and leads to defects in nodulation of alfalfa plants. In this study, we found that S. meliloti fadD mutants accumulated a mixture of free fatty acids during the stationary phase of growth. The composition of the free fatty acid pool and the results obtained after specific labeling of esterified fatty acids with a Δ5-desaturase (Δ5-Des) were in agreement with membrane phospholipids being the origin of the released fatty acids. Escherichia coli fadD mutants also accumulated free fatty acids released from membrane lipids in the stationary phase. This phenomenon did not occur in a mutant of E. coli with a deficient FadL fatty acid transporter, suggesting that the accumulation of fatty acids in fadD mutants occurs inside the cell. Our results indicate that, besides the activation of exogenous LCFA, in bacteria FadD plays a major role in the activation of endogenous fatty acids released from membrane lipids. Furthermore, expression analysis performed with S. meliloti revealed that a functional FadD is required for the upregulation of genes involved in fatty acid degradation and suggested that in the wild-type strain, the fatty acids released from membrane lipids are degraded by β-oxidation in the stationary phase of growth.