Disruption and analysis of the clpB, clpC, and clpE genes in Lactococcus lactis: ClpE, a new Clp family in gram-positive bacteria

In the genome of the gram-positive bacterium Lactococcus lactis MG1363, we have identified three genes (clpC, clpE, and clpB) which encode Clp proteins containing two conserved ATP binding domains. The proteins encoded by two of the genes belong to the previously described ClpB and ClpC families. The clpE gene, however, encodes a member of a new Clp protein family that is characterized by a short N-terminal domain including a putative zinc binding domain (-CX2CX22CX2C-). Expression of the 83-kDa ClpE protein as well as of the two proteins encoded by clpB was strongly induced by heat shock and, while clpC mRNA synthesis was moderately induced by heat, we were unable to identify the ClpC protein. When we analyzed mutants with disruptions in clpB, clpC, or clpE, we found that although the genes are part of the L. lactis heat shock stimulon, the mutants responded like wild-type cells to heat and salt treatments. However, when exposed to puromycin, a tRNA analogue that results in the synthesis of truncated, randomly folded proteins, clpE mutant cells formed smaller colonies than wild-type cells and clpB and clpC mutant cells. Thus, our data suggest that ClpE, along with ClpP, which recently was shown to participate in the degradation of randomly folded proteins in L. lactis, could be necessary for degrading proteins generated by certain types of stress.     

Induction of the heat shock protein ClpB affects cold acclimation in the cyanobacterium Synechococcus sp. strain PCC 7942.

The heat shock protein ClpB is essential for acquired thermotolerance in cyanobacteria and eukaryotes and belongs to a diverse group of polypeptides which function as molecular chaperones. In this study we show that ClpB is also strongly induced during moderate cold stress in the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. A fivefold increase in ClpB (92 kDa) content occurred when cells were acclimated to 25 degrees C over 24 h after being shifted from the optimal growth temperature of 37 degrees C. A corresponding increase occurred for the smaller ClpB' (78 kDa), which arises from a second translational start within the clpB gene of prokaryotes. Shifts to more extreme cold (i.e., 20 and 15 degrees C) progressively decreased the level of ClpB induction, presumably due to retardation of protein synthesis within this relatively cold-sensitive strain. Inactivation of clpB in Synechococcus sp. increased the extent of inhibition of photosynthesis upon the shift to 25 degrees C and markedly reduced the mutant's ability to acclimate to the new temperature regime, with a threefold drop in growth rate. Furthermore, around 30% fewer delta clpB cells survived the shift to 25 degrees C after 24 h compared to the wild type, and more of the mutant cells were also arrested during cell division at 25 degrees C, remaining attached after septum formation. Development of a cold thermotolerance assay based on cell survival clearly demonstrated that wild-type cells could acquire substantial resistance to the nonpermissive temperature of 15 degrees C by being pre-exposed to 25 degrees C. The same level of cold thermotolerance, however, occurred in the delta clpB strain, indicating ClpB induction is not necessary for this form of thermal resistance in Synechococcus spp. Overall, our results demonstrate that the induction of ClpB contributes significantly to the acclimation process of cyanobacteria to permissive low temperatures.     

The heat shock protein ClpB mediates the development of thermotolerance in the cyanobacterium Synechococcus sp. strain PCC 7942.

The heat shock protein CIpB (HSP100) is a member of the diverse group of Clp polypeptides that function as molecular chaperones and/or regulators of energy-dependent proteolysis. A single-copy gene coding for a ClpB homolog was cloned and sequenced from the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. The predicted polypeptide sequence was most similar to sequences of cytosolic ClpB from bacteria and higher plants (i.e., 70 to 75%). Inactivation of clpB in Synechococcus sp. strain PCC 7942 resulted in no significant differences from the wild-type phenotype under optimal growth conditions. In the wild type, two forms of ClpB were induced during temperature shifts from 37 to 47.5 or 50 degrees C, one of 92 kDa, which matched the predicted size, and another smaller protein of 78 kDa. Both proteins were absent in the delta clpB strain. The level of induction of the two ClpB forms in the wild type increased with increasingly higher temperatures, while the level of the constitutive ClpC protein remained unchanged. In the delta clpB strain, however, the ClpC content almost doubled during the heating period, presumably to compensate for the loss of ClpB activity. Photosynthetic measurements at 47.5 and 50 degrees C showed that the null mutant was no more susceptible to thermal inactivation than the wild type. Using photosynthesis as a metabolic indicator, an assay was developed for Synechococcus spp. to determine the importance of ClpB for acquired thermotolerance. Complete inactivation of photosynthetic oxygen evolution occurred in both the wild type and the delta clpB strain when they were shifted from 37 directly to 55 degrees C for 10 min. By preexposing the cells at 50 degrees C for 1.5 h, however, a significant level of photosynthesis was retained in the wild type but not in the mutant after the treatment at 55 degrees C for 10 min. Cell survival determinations confirmed that the loss of ClpB synthesis caused a fivefold reduction in the ability of Synechococcus cells to develop thermotolerance. These results clearly show that induction of ClpB at high temperatures is vital for sustained thermotolerance in Synechococcus spp., the first such example for either a photosynthetic or a prokaryotic organism.     

Enhanced biofilm formation and loss of capsule synthesis: deletion of a putative glycosyltransferase in Porphyromonas gingivalis

Periodontitis is a biofilm-mediated disease. Porphyromonas gingivalis is an obligate anaerobe consistently associated with severe manifestations of this disease. As an opportunistic pathogen, the ability to proliferate within and disseminate from subgingival biofilm (plaque) is central to its virulence. Here, we report the isolation of a P. gingivalis transposon insertion mutant altered in biofilm development and the reconstruction and characterization of this mutation in three different wild-type strains. The mutation responsible for the altered biofilm phenotype was in a gene with high sequence similarity ( approximately 61%) to a glycosyltransferase gene. The gene is located in a region of the chromosome that includes up to 16 genes predicted to be involved in the synthesis and transport of capsular polysaccharide. The phenotype of the reconstructed mutation in all three wild-type backgrounds is that of enhanced biofilm formation. In addition, in strain W83, a strain that is encapsulated, the glycosyltransferase mutation resulted in a loss of capsule. Further experiments showed that the W83 mutant strain was more hydrophobic and exhibited increased auto-aggregation. Our results indicate that we have identified a gene involved in capsular-polysaccharide synthesis in P. gingivalis and that the production of capsule prevented attachment and the initiation of in vitro biofilm formation on polystyrene microtiter plates.     

Cloning and characterization of two cellulase genes from Lentinula edodes

The effect of overproduction of the Hsp70 system proteins (DnaK, DnaJ, GrpE) and/or ClpB (Hsp100) from plasmids on the process of formation and removal of heat-aggregated proteins from Escherichia coli cells (the S fraction) was investigated by sucrose density gradient centrifugation. Two plasmids were employed: pKJE7 carrying the dnaK/dnaJ/grpE genes under the control of the araB promoter and pClpB carrying the clpB gene under the control of its own promoter (sigma(32)-dependent). In the wild-type cells the S fraction after 15 min of heat shock amounted to 21% of cellular insoluble proteins (IP), and disappeared 10 min after transfer of the culture to 37 degrees C. In contrast to this, in the clpB mutant the S fraction was larger (35% IP) and its elimination was retarded, nearly 60% of the aggregated proteins remained stable 30 min after heat shock. This result points to the importance of ClpB in removal of the heat-aggregated proteins from cells. Overproduction of the Hsp70 system proteins (exceeding by about 1.5-fold that of wild-type) in wild-type and DeltaclpB cells completely prevented the formation of the S fraction during heat shock. Overproduction of ClpB (exceeding by about eight-fold that of wild-type) in the same background did not prevent protein aggregation after heat shock and only partly compensated for the effect of the mutation in the clpB gene. Monitoring the S fraction during co-production of DnaK/DnaJ/GrpE and ClpB in the DeltaclpB mutant revealed that both the levels of expression and the ratios of ClpB to Hsp70 system proteins had a significant effect on the formation and removal of protein aggregates in heat-shocked E. coli cells. In the presence of excess ClpB, an increase in the levels of DnaK, DnaJ and GrpE was required to prevent aggregate formation upon heat shock or to efficiently remove protein aggregates after heat shock. Therefore, it is supposed that a high level of ClpB under some conditions, especially at insufficient levels of Hsp70 system proteins, may support protein aggregation resulting from heat shock and may lead to stabilization of hydrophobic aggregates.     

Characterization of Brucella suis clpB and clpAB mutants and participation of the genes in stress responses

Pathogens often encounter stressful conditions inside their hosts. In the attempt to characterize the stress response in Brucella suis, a gene highly homologous to Escherichia coli clpB was isolated from Brucella suis, and the deduced amino acid sequence showed features typical of the ClpB ATPase family of stress response proteins. Under high-temperature stress conditions, ClpB of B. suis was induced, and an isogenic B. suis clpB mutant showed increased sensitivity to high temperature, but also to ethanol stress and acid pH. The effects were reversible by complementation. Simultaneous inactivation of clpA and clpB resulted in a mutant that was sensitive to oxidative stress. In B. suis expressing gfp, ClpA but not ClpB participated in degradation of the green fluorescent protein at 42 degrees C. We concluded that ClpB was responsible for tolerance to several stresses and that the lethality caused by harsh environmental conditions may have similar molecular origins.     

The Escherichia coli heat shock protein ClpB restores acquired thermotolerance to a cyanobacterial clpB deletion mutant

In both prokaryotes and eukaryotes, the heat shock protein ClpB functions as a molecular chaperone and plays a key role in resisting high temperature stress. ClpB is important for the development of thermotolerance in yeast and cyanobacteria but apparently not in Escherichia coli. We undertook a complementation study to investigate whether the ClpB protein from E coli (EcClpB) differs functionally from its cyanobacterial counterpart in the unicellular cyanobacterium Synechococcus sp. PCC 7942. The EcClpB protein is 56% identical to its ClpB1 homologue in Synechococcus. A plasmid construct was prepared containing the entire E coli clpB gene under the control of the Synechococcus clpB1 promoter. This construct was transformed into a Synechococcus clpB1 deletion strain (deltaclpB1) and integrated into a phenotypically neutral site of the chromosome. The full-length EcClpB protein (EcClpB-93) was induced in the transformed Synechococcus strain during heat shock as well as the smaller protein (EcClpB-79) that arises from a second translational start inside the single clpB message. Using cell survival measurements we show that the EcClpB protein can complement the Synechococcus deltaclpB1 mutant and restore its ability to develop thermotolerance. We also demonstrate that both EcClpB-93 and -79 appear to contribute to the degree of acquired thermotolerance restored to the Synechococcus complementation strains.     

Small heat shock proteins, ClpB and the DnaK system form a functional triade in reversing protein aggregation

Small heat shock proteins (sHsps) can efficiently prevent the aggregation of unfolded proteins in vitro. However, how this in vitro activity translates to function in vivo is poorly understood. We demonstrate that sHsps of Escherichia coli, IbpA and IbpB, co-operate with ClpB and the DnaK system in vitro and in vivo, forming a functional triade of chaperones. IbpA/IbpB and ClpB support independently and co-operatively the DnaK system in reversing protein aggregation. A delta ibpAB delta clpB double mutant exhibits strongly increased protein aggregation at 42 degrees C compared with the single mutants. sHsp and ClpB function become essential for cell viability at 37 degrees C if DnaK levels are reduced. The DnaK requirement for growth is increasingly higher for delta ibpAB, delta clpB, and the double delta ibpAB delta clpB mutant cells, establishing the positions of sHsps and ClpB in this chaperone triade.     

Proinflammatory gene expression in mouse ST2 cell line in response to infection by Porphyromonas gingivalis

Porphyromonas gingivalis is a predominant periodontal pathogen, whose infection causes inflammatory responses in periodontal tissue and alveolar bone resorption. Various virulence factors of this pathogen modulate host innate immune responses. It has been reported that gingipains degrade a wide variety of host cell proteins, and fimbriae are involved in bacterial adhesion to and invasion of host cells. In the present study, we profiled ST2 stromal cell gene expression following infection with the viable P. gingivalis strain ATCC33277 as well as with its gingipain- and fimbriae-deficient mutants, using microarray technology and quantitative real-time polymerase chain reaction. Using a mouse array of about 20,000 genes, we found that infection with the wild strain elicited a significant upregulation (greater than 2-fold) of expression of about 360 genes in ST2 cells, which included the chemokines CCL2, CCL5, and CXCL10, and other proinflammatory proteins such as interleukin-6 (IL-6) and matrix metalloproteinase-13 (MMP-13). Further, infection with the gingipain-deficient mutant elicited a reduced expression of the CXCL10, IL-6 and MMP-13 genes, suggesting that gingipains play an important role in inducing the expression of those genes following P. gingivalis infection. On the other hand, the pattern of global gene expression induced by the fimbriae-deficient mutant was similar to that by the wild strain. These results suggest that P. gingivalis infection induces gene expression of a wide variety of proinflammatory proteins in stromal cells/osteoblasts, and gingipains may be involved in inducing several of the proinflammatory factors.     

Porphyromonas gingivalis Strain Specific Interactions with Human Coronary Artery Endothelial Cells: A Comparative Study

Both epidemiologic and experimental findings suggest that infection with Porphyromonas gingivalis exacerbates progression of atherosclerosis. As P. gingivalis exhibits significant strain variation, it is reasonable that different strains possess different capabilities and/or mechanisms by which they promote atherosclerosis. Using P. gingivalis strains that have been previously evaluated in the ApoE null atherosclerosis model, we assessed the ability of W83, A7436, 381, and 33277 to adhere, invade, and persist in human coronary artery endothelial (HCAE) cells. W83 and 381 displayed an equivalent ability to adhere to HCAE cells, which was significantly greater than both A7436 and 33277 (P<0.01). W83, 381, and 33277 were more invasive than A7436 (P<0.0001). However, only W83 and A7436 were able to remain viable up to 48 hours in HCAE cell cultures, whereas 381 was cleared by 48 hours and 33277 was cleared by 24 hours. These differences in persistence were in part due to strain specific differences in intracellular trafficking. Both W83 and 381 trafficked through the autophagic pathway, but not A7436 or 33277. Internalized 381 was the only strain that was dependent upon the autophagic pathway for its survival. Finally, we assessed the efficacy of these strains to activate HCAE cells as defined by production of IL-6, IL-8, IL-12p40, MCP-1, RANTES, TNF-α, and soluble adhesion molecules (sICAM-1, sVCAM-1, and sE-selectin). Only moderate inflammation was observed in cells infected with either W83 or A7436, whereas cells infected with 381 exhibited the most profound inflammation, followed by cells infected with 33277. These results demonstrate that virulence mechanisms among different P. gingivalis strains are varied and that pathogenic mechanisms identified for one strain are not necessarily applicable to other strains.     

The truncated form of the bacterial heat shock protein ClpB/HSP100 contributes to development of thermotolerance in the cyanobacterium Synechococcus sp. strain PCC 7942

ClpB is a highly conserved heat shock protein that is essential for thermotolerance in bacteria and eukaryotes. One distinctive feature of all bacterial clpB genes is the dual translation of a truncated 79-kDa form (ClpB-79) in addition to the full-length 93-kDa protein (ClpB-93). To investigate the currently unknown function of ClpB-79, we have examined the ability of the two different-sized ClpB homologues from the cyanobacterium Synechococcus sp. strain PCC 7942 to confer thermotolerance. We show that the ClpB-79 form has the same capacity as ClpB-93 to confer thermotolerance and that the ClpB-79 protein contributes ca. one-third of the total thermotolerance developed in wild-type Synechococcus, the first in vivo demonstration of a functional role for ClpB-79 in bacteria.     

Involvement of integrins in fimbriae-mediated binding and invasion by Porphyromonas gingivalis

Interaction between the major fimbriae of Porphyromonas gingivalis and gingival epithelial cells is important for bacterial adhesion and invasion. In this study, we identified integrins as an epithelial cell cognate receptor for P. gingivalis fimbriae. Immunoprecipitation and direct binding assays revealed a physical association between recombinant fimbrillin and beta1 integrins. In vitro adhesion and invasion assays demonstrated inhibition of binding and invasion of P. gingivalis by beta1 integrin antibodies. In contrast, invasion of a fimbriae-deficient mutant of P. gingivalis was not affected by integrin antibodies. Infection of gingival epithelial cells with wild-type P. gingivalis induced tyrosine phosphorylation of the 68 kDa focal adhesion protein paxillin, whereas the fimbriae-deficient mutant failed to evoke similar changes. Interestingly, activation of paxillin was not accompanied by an increase in the phosphorylation of focal adhesion kinase (FAK). These results provide evidence that P. gingivalis fimbriae promote adhesion to gingival epithelial cells through interaction with beta1 integrins, and this association represents a key step in the induction of the invasive process and subsequent cell responses to P. gingivalis infection.