The htrA (degP) gene of Listeria monocytogenes 10403S is essential for optimal growth under stress conditions

This report describes a mutant of Listeria monocytogenes strain 10403S (serotype 1/2a) with a defective response to conditions of high osmolarity, an environment that L. monocytogenes encounters in some ready-to-eat foods. A library of L. monocytogenes clones mutagenized with Tn917 was generated and scored for sensitivity to 4% NaCl in order to identify genes responsible for growth or survival in elevated-NaCl environments. One of the L. monocytogenes Tn917 mutants, designated strain OSM1, was selected, and the gene interrupted by the transposon was sequenced. A BLAST search with the putative translated amino acid sequence indicated that the interrupted gene product was a homolog of htrA (degP), a gene coding for a serine protease identified as a stress response protein in several gram-positive and gram-negative bacteria. An htrA deletion strain, strain LDW1, was constructed, and the salt-sensitive phenotype of this strain was complemented by introduction of a plasmid carrying the wild-type htrA gene, demonstrating that htrA is necessary for optimal growth under conditions of osmotic stress. Additionally, strain LDW1 was tested for its response to temperature and H(2)O(2) stresses. The results of these growth assays indicated that strain LDW1 grew at a lower rate than the wild-type strain at 44 degrees C but at a rate similar to that of the wild-type strain when incubated at 4 degrees C. In addition, strain LDW1 was significantly more sensitive to a 52 degrees C heat shock than the wild-type strain. Strain LDW1 was also defective in its response to H(2)O(2) challenge at 37 degrees C, since 100 or 150 micro g of H(2)O(2) was more inhibitory for the growth of strain LDW1 than for that of the parent strain. The stress response phenotype observed for strain LDW1 is similar to that observed for other HtrA(-) organisms, which suggests that L. monocytogenes HtrA may play a role in degrading misfolded proteins that accumulate under stress conditions.     

Expression of the Staphylococcus aureus surface proteins HtrA1 and HtrA2 in Lactococcus lactis

Staphylococcus aureus encodes two HtrA-like serine surface proteases, called HtrA1 and HtrA2. The roles of these HtrA homologs were distinguished by expression studies in a heterologous host, Lactococcus lactis, whose single extracellular protease, HtrA(Ll), was absent. HtrA(Ll) is involved in stress resistance, and processing and/or degradation of extracellular proteins. Controlled expression of staphylococcal htrA1 and htrA2 was achieved in L. lactis strain NZ9000 DeltahtrA, as confirmed with anti-HtrA1 and anti-HtrA2 specific antibodies. HtrA1 fully restored thermo-resistance to the htrA-defective L. lactis strain, despite a poor capacity to degrade abnormal or truncated proteins. We therefore propose that activities of HtrA1 other than proteolysis may be sufficient for high-temperature growth complementation. HtrA2 is 36% identical to HtrA(Ll), and was highly expressed in L. lactis; nevertheless, it displayed nearly no detectable activities. The poor proteolytic activities of staphylococcal HtrA proteins in L. lactis may reflect a requirement for S. aureus-specific co-factors.     

Investigation into the role of the serine protease HtrA in Yersinia pestis pathogenesis

The HtrA stress response protein has been shown to play a role in the virulence of a number of pathogens. For some organisms, htrA mutants are attenuated in the animal model and can be used as live vaccines. A Yersinia pestis htrA orthologue was identified, cloned and sequenced, showing 86% and 87% similarity to Escherichia coli and Salmonella typhimurium HtrAs. An isogenic Y. pestis htrA mutant was constructed using a reverse genetics approach. In contrast to the wild-type strain, the mutant failed to grow at an elevated temperature of 39 degrees C, but showed only a small increase in sensitivity to oxidative stress and was only partially attenuated in the animal model. However, the mutant exhibited a different protein expression profile to that of the wild-type strain when grown at 28 degrees C to simulate growth in the flea.     

The HtrA protease of Campylobacter jejuni is required for heat and oxygen tolerance and for optimal interaction with human epithelial cells

Campylobacter jejuni is a predominant cause of food-borne bacterial gastroenteritis in the developed world. We have investigated the importance of a homologue of the periplasmic HtrA protease in C. jejuni stress tolerance. A C. jejuni htrA mutant was constructed and compared to the parental strain, and we found that growth of the mutant was severely impaired both at 44 degrees C and in the presence of the tRNA analogue puromycin. Under both conditions, the level of misfolded protein is known to increase, and we propose that the heat-sensitive phenotype of the htrA mutant is caused by an accumulation of misfolded protein in the periplasm. Interestingly, we observed that the level of the molecular chaperones DnaK and ClpB was increased in the htrA mutant, suggesting that accumulation of non-native proteins in the periplasm induces the expression of cytoplasmic chaperones. While lack of HtrA reduces the oxygen tolerance of C. jejuni, the htrA mutant was not sensitive to compounds that increase the formation of oxygen radicals, such as paraquat, cumene hydroperoxide, and H2O2. Using tissue cultures of human epithelial cells (INT407), we found that the htrA mutant adhered to and invaded human epithelial cells with a decreased frequency compared to the wild-type strain. This defect may be a consequence of the observed altered morphology of the htrA mutant. Thus, our results suggest that in C. jejuni, HtrA is important for growth during stressful conditions and has an impact on virulence.     

Physiological role of beta-phosphoglucomutase in Lactococcus lactis

A beta-phosphoglucomutase (beta-PGM) mutant of Lactococcus lactis subsp. lactis ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of beta-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h(-1), while the deletion of beta-PGM resulted in a maximum specific growth rate of 0.05 h(-1) on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as beta-glucose 1-phosphate in the medium. Furthermore, the beta-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of alpha-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the beta-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded beta-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in L. lactis.     

HtrA is the unique surface housekeeping protease in Lactococcus lactis and is required for natural protein processing

We identified an exported protease in Lactococcus lactis ssp. lactis strain IL1403 belonging to the HtrA/DegP family. Inactivation of the chromosomal gene (htrALl) encoding this protease (HtrALl) results in growth thermo-sensitivity at very high temperatures (above 37 degrees C for L. lactis). The role of htrALl in extracellular proteolysis under normal growth conditions was examined by testing the stability of different exported proteins (i.e. fusions, a heterologous pre-pro-protein or a native protein containing repeats), having different locations. In the wild-type (wt) strain, degradation products, including the C-terminal protein ends, were present in the medium, indicating that proteolysis occurs during or after export to the cell surface; in one case, degradation was nearly total. In contrast, proteolysis was totally abolished in the htrA strain for all five proteins tested, and the yield of full-length products was significantly increased. These results suggest that HtrALl is the sole extracellular protease that degrades abnormal exported proteins. In addition, our results reveal that HtrALl is needed for the pro-peptide processing of a natural pro-protein and for maturation of a native protein. We propose that in lactococci, and possibly in other Gram-positive organisms with small sized-genomes, a single surface protease, HtrA, is totally responsible for the housekeeping of exported proteins.     

Glyceraldehyde-3-phosphate dehydrogenase regulation in Lactococcus lactis ssp. cremoris MG1363 or relA mutant at low pH

AIMS: To analyse the phenotype of a relA acid-resistant mutant of Lactococcus lactis ssp. cremoris MG1363, and to compare the glyceraldehyde-3-phosphate dehydrogenase regulation in both strains. METHODS AND RESULTS: Lactococcus lactis ssp. cremoris MG1363 and the relA mutant affected in the (p)ppGpp synthetase were grown in a series of batch-mode fermentation at different pH-regulated conditions with glucose as carbon substrate. All the determinants of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) regulation were quantified. In L. lactis MG1363, the GAPDH was strongly inhibited in vitro by decreased pH values, but this inhibition was totally compensated in vivo by the lower NADH/NAD+ ratio and more efficiently by the important increase in the intracellular amount of GAPDH. In contrast to the wild type, GAPDH activity of the relA strain was not increased when grown at low pH but the level of GAPDH remained constitutively high. However, pH homeostasis was not improved in the relA mutant and it grew slower and exhibited a lower glycolytic flux than the wild-type strain at low pH. CONCLUSIONS: Despite a better resistance to acid stress, the increased survival in L. lactis relA mutant at low pH was not related with an improved pH homeostasis but was associated with a diminished capacity to maintain a high flux through glycolysis. SIGNIFICANCE AND IMPACT OF THE STUDY: The phenotype of a strong acid-resistant L. lactis strain was established in acid conditions and some key metabolic parameters compared with the wild type. This analysis led to the conclusion that growth and survival seem to be antinomic parameters, since improving one of them leads to a decrease in the other one.     

Involvement of the reserve material poly-beta-hydroxybutyrate in Azospirillum brasilense stress endurance and root colonization

When grown under suboptimal conditions, rhizobacteria of the genus Azospirillum produce high levels of poly-beta-hydroxybutyrate (PHB). Azospirillum brasilense strain Sp7 and a phbC (PHB synthase) mutant strain in which PHB production is impaired were evaluated for metabolic versatility, for the ability to endure various stress conditions, for survival in soil inoculants, and for the potential to promote plant growth. The carbon source utilization data were similar for the wild-type and mutant strains, but the generation time of the wild-type strain was shorter than that of the mutant strain with all carbon sources tested. The ability of the wild type to endure UV irradiation, heat, osmotic pressure, osmotic shock, and desiccation and to grow in the presence of hydrogen peroxide was greater than that of the mutant strain. The motility and cell aggregation of the mutant strain were greater than the motility and cell aggregation of the wild type. However, the wild type exhibited greater chemotactic responses towards attractants than the mutant strain exhibited. The wild-type strain exhibited better survival than the mutant strain in carrier materials used for soil inoculants, but no difference in the ability to promote plant growth was detected between the strains. In soil, the two strains colonized roots to the same extent. It appears that synthesis and utilization of PHB as a carbon and energy source by A. brasilense under stress conditions favor establishment of this bacterium and its survival in competitive environments. However, in A. brasilense, PHB production does not seem to provide an advantage in root colonization under the conditions tested.     

Involvement of the Reserve Material Poly-β-Hydroxybutyrate in Azospirillum brasilense Stress Endurance and Root Colonization

When grown under suboptimal conditions, rhizobacteria of the genus Azospirillum produce high levels of poly-β-hydroxybutyrate (PHB). Azospirillum brasilense strain Sp7 and a phbC (PHB synthase) mutant strain in which PHB production is impaired were evaluated for metabolic versatility, for the ability to endure various stress conditions, for survival in soil inoculants, and for the potential to promote plant growth. The carbon source utilization data were similar for the wild-type and mutant strains, but the generation time of the wild-type strain was shorter than that of the mutant strain with all carbon sources tested. The ability of the wild type to endure UV irradiation, heat, osmotic pressure, osmotic shock, and desiccation and to grow in the presence of hydrogen peroxide was greater than that of the mutant strain. The motility and cell aggregation of the mutant strain were greater than the motility and cell aggregation of the wild type. However, the wild type exhibited greater chemotactic responses towards attractants than the mutant strain exhibited. The wild-type strain exhibited better survival than the mutant strain in carrier materials used for soil inoculants, but no difference in the ability to promote plant growth was detected between the strains. In soil, the two strains colonized roots to the same extent. It appears that synthesis and utilization of PHB as a carbon and energy source by A. brasilense under stress conditions favor establishment of this bacterium and its survival in competitive environments. However, in A. brasilense, PHB production does not seem to provide an advantage in root colonization under the conditions tested.     

A respiratory-deficient mutation associated with high salt sensitivity in Kluyveromyces lactis

A salt-sensitive mutant of Kluyveromyces lactis was isolated that was unable to grow in high-salt media. This mutant was also respiratory-deficient and temperature-sensitive for growth. The mutation mapped in a single nuclear gene that is the ortholog of BCS1 of Saccharomyces cerevisiae. The BCS1 product is a mitochondrial protein required for the assembly of respiratory complex III. The bcs1 mutation of S. cerevisiae leads to a loss of respiration, but, unlike in K. lactis, it is not accompanied by salt sensitivity. All the respiratory-deficient K. lactis mutants tested were found to be salt-sensitive compared to their isogenic wild-type strains. In the presence of the respiratory inhibitor antimycin A, the wild-type strain also became salt-sensitive. By contrast, none of the S. cerevisiae respiratory-deficient mutants tested showed increased salt sensitivity. The salt sensitivity of the Klbcs1 mutant, but not its respiratory deficiency, was suppressed by the multicopy KlVMA13 gene, a homolog of the S. cerevisiae VMA13 gene encoding a subunit of the vacuolar H(+)-ATPase. These results suggest that cellular salt homeostasis in K. lactis is strongly dependent on mitochondrial respiratory activity, and/or that the ion homeostasis of mitochondria themselves could be a primary target of salt stress.     

Function of Trehalose and Glycogen in Cell Cycle Progression and Cell Viability in Saccharomyces cerevisiae

Trehalose and glycogen accumulate in Saccharomyces cerevisiae when growth conditions deteriorate. It has been suggested that aside from functioning as storage factors and stress protectants, these carbohydrates may be required for cell cycle progression at low growth rates under carbon limitation. By using a mutant unable to synthesize trehalose and glycogen, we have investigated this requirement of trehalose and glycogen under carbon-limited conditions in continuous cultures. Trehalose and glycogen levels increased with decreasing growth rates in the wild-type strain, whereas no trehalose or glycogen was detected in the mutant. However, the mutant was still able to grow and divide at low growth rates with doubling times similar to those for the wild-type strain, indicating that trehalose and glycogen are not essential for cell cycle progression. Nevertheless, upon a slight increase of extracellular carbohydrates, the wild-type strain degraded its reserve carbohydrates and was able to enter a cell division cycle faster than the mutant. In addition, wild-type cells survived much longer than the mutant cells when extracellular carbon was exhausted. Thus, trehalose and glycogen have a dual role under these conditions, serving as storage factors during carbon starvation and providing quickly a higher carbon and ATP flux when conditions improve. Interestingly, the CO₂ production rate and hence the ATP flux were higher in the mutant than in the wild-type strain at low growth rates. The possibility that the mutant strain requires this steady higher glycolytic flux at low growth rates for passage through Start is discussed.     

HtrA is a major virulence determinant of Bacillus anthracis

We demonstrate that disruption of the htrA (high temperature requirement A) gene in either the virulent Bacillus anthracis Vollum (pXO1(+) , pXO2(+) ), or in the ΔVollum (pXO1(-), pXO2(-), nontoxinogenic and noncapsular) strains, affect significantly the ability of the resulting mutants to withstand heat, oxidative, ethanol and osmotic stress. The ΔhtrA mutants manifest altered secretion of several proteins, as well as complete silencing of the abundant extracellular starvation-associated neutral protease A (NprA). VollumΔhtrA bacteria exhibit delayed proliferation in a macrophage infection assay, and despite their ability to synthesize the major B. anthracis toxins LT (lethal toxin) and ET (oedema toxin) as well as the capsule, show a decrease of over six orders of magnitude in virulence (lethal dose 50% = 3 × 10(8) spores, in the guinea pig model of anthrax), as compared with the parental wild-type strain. This unprecedented extent of loss of virulence in B. anthracis, as a consequence of deletion of a single gene, as well as all other phenotypic defects associated with htrA mutation, are restored in their corresponding trans-complemented strains. It is suggested that the loss of virulence is due to increased susceptibility of the ΔhtrA bacteria to stress insults encountered in the host. On a practical note, it is demonstrated that the attenuated Vollum ΔhtrA is highly efficacious in protecting guinea pigs against a lethal anthrax challenge.     

Glucose/citrate cometabolism in Lactococcus lactis subsp. lactis biovar diacetylactis with impaired alpha-acetolactate decarboxylase

The pyruvate metabolism of a Lactococcus lactis subsp. lactis biovar diacetylactis mutant deficient in alpha-acetolactate decarboxylase and its wild-type strain was studied during batch cultivations. A chemically defined medium was used containing glucose as carbon- and energy-source. The alpha-acetolactate decarboxylase deficiency had no effect on the specific growth rate. Addition of citrate was found to increase the specific growth rate of both strains under aerobic and anaerobic conditions. The product formation was monitored throughout the cultivations. The carbon- and redox-balances were within the accuracy of the experimental data. When citrate was added, alpha-acetolactate, diacetyl, and acetoin were formed, and aeration was shown to have a positive effect on the formation of these metabolites. By omitting lipoic acid (required for a functional pyruvate dehydrogenase complex) from the growth medium, a similar stimulatory effect on alpha-acetolactate, diacetyl, and acetoin formation was observed under aerobic conditions. The strain with impaired alpha-acetolactate decarboxylase activity accumulated alpha-acetolactate which resulted in an increased diacetyl formation compared to the wild-type strain, under aerobic and anaerobic conditions.     

Regulatory functions of serine-46-phosphorylated HPr in Lactococcus lactis

In most low-G+C gram-positive bacteria, the phosphoryl carrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) becomes phosphorylated at Ser-46. This ATP-dependent reaction is catalyzed by the bifunctional HPr kinase/P-Ser-HPr phosphatase. We found that serine-phosphorylated HPr (P-Ser-HPr) of Lactococcus lactis participates not only in carbon catabolite repression of an operon encoding a beta-glucoside-specific EII and a 6-P-beta-glucosidase but also in inducer exclusion of the non-PTS carbohydrates maltose and ribose. In a wild-type strain, transport of these non-PTS carbohydrates is strongly inhibited by the presence of glucose, whereas in a ptsH1 mutant, in which Ser-46 of HPr is replaced with an alanine, glucose had lost its inhibitory effect. In vitro experiments carried out with L. lactis vesicles had suggested that P-Ser-HPr is also implicated in inducer expulsion of nonmetabolizable homologues of PTS sugars, such as methyl beta-D-thiogalactoside (TMG) and 2-deoxy-D-glucose (2-DG). In vivo experiments with the ptsH1 mutant established that P-Ser-HPr is not necessary for inducer expulsion. Glucose-activated 2-DG expulsion occurred at similar rates in wild-type and ptsH1 mutant strains, whereas TMG expulsion was slowed in the ptsH1 mutant. It therefore seems that P-Ser-HPr is not essential for inducer expulsion but that in certain cases it can play an indirect role in this regulatory process.     

弗氏2a志贺菌htrA基因突变体的构建

目的:构建弗氏2a志贺菌2457T的htrA基因缺失突变株及HtrA酶失活突变株,以便进一步研究HtrA蛋白的功能。方法:用PCR扩增htrA基因上下游同源臂,构建含有kan基因的打靶片段,采用λ-Red重组系统对htrA基因进行缺失,用PCR进行验证;通过定点突变的方法构建HtrA酶失活突变株,并测序验证。结果与结论:构建了2457T htrA缺失突变株和2457T/htrAFSA酶失活突变株。     

Effects of metabolic flux on stress response pathways in Lactococcus lactis

Studies of cellular responses to stress conditions such as heat, oxygen or starvation have revealed the existence of numerous specific or interactive response pathways. We previously observed in Lactococcus lactis that inactivation of the recA gene renders the lactococcal strain sensitive not only to DNA-damaging agents but also to oxygen and heat. To further examine the stress response pathways in L. lactis, we isolated thermoresistant insertional mutants (Trm) of the recA strain. Eighteen independent trm mutations were identified and characterized. We found that mutations map in only seven genes, implicated in purine metabolism (deoB, guaA and tktA), phosphate uptake (pstB and pstS), mRNA stability (pnpA) and in one uncharacterized gene (trmA). All the trm mutations, with the exception of trmA, confer multiple stress resistance to the cell. Some of the mutations confer improved heat stress resistance not only in the recA but also in the wild-type context. Our results reveal that cellular metabolic pathways are intimately related to stress response and that the flux of particular metabolites, notably guanine and phosphate, may be implicated in stress response in lactococci.     

A membrane protein is required for bacteriophage c2 infection of Lactococcus lactis subsp. lactis C2.

Phage-resistant mutants, isolated from cultures of Lactococcus lactis subsp. lactis C2 infected with phage c2, did not form plaques but bound phage normally. The mutants were sensitive to another phage, sk1, although the number of plaques was reduced approximately 56% and the plaques were four times smaller. Binding to phage sk1 was reduced about 10%. Another group of phage-resistant mutants, isolated from cultures infected with phage sk1, bound normally to both phages c2 and sk1 but did not form plaques with either phage. Carbohydrate analyses by gas chromatography of the cell walls showed no significant differences in saccharide compositions between the wild-type and phage-resistant cells. However, a difference was observed in the interactions of the phage with the cytoplasmic membranes. Membranes from the wild-type cells, but not mutant cells, inactivated phage c2. Phage sk1 was not inactivated by membrane from either strain. Treatment of wild-type membranes with proteinase K eliminated the ability of the membrane to inactivate the phage, whereas treatment with mutanolysin had no effect. On the basis of this ability to inactivate the phage, a membrane protein was partially purified by gel filtration and ion-exchange chromatography. Under nondenaturing conditions, the phage-inactivating protein has an apparent Mr of approximately 350,000. The protein has an apparent subunit size of 32 kDa, which suggests that it normally exists as a multimer with 10 to 12 subunits or in association with other membrane components. It is proposed that this protein is required for phage c2 infection.