A role for Asp75 in domain interactions in the Bacillus subtilis response regulator Spo0A.

Spo0A is a two-domain response regulator required for sporulation initiation in Bacillus subtilis. Studies on response regulators have focused on the activity of each domain, but very little is known about the mechanism by which the regulatory domain inhibits the activator domain. In this study, we created a single amino acid substitution in the regulatory domain, D75S, which resulted in a dramatic decrease in sporulation in vivo. In vitro studies with the purified Spo0AD75S protein demonstrated that phosphorylation and DNA binding were comparable with wild type Spo0A. However, the mutant was unable to stimulate transcription by final sigma(A)-RNA polymerase from the Spo0A-dependent spoIIG operon promoter. We suggest that the amino acid Asp(75) and/or the region within which it resides, the alpha3-beta4 loop, are involved in the inhibitory interaction between the regulatory and activator domains of Spo0A.

The Spo0A sof mutations reveal regions of the regulatory domain that interact with a sensor kinase and RNA polymerase.

Spo0A is a two-domain response regulator required for the initiation of sporulation in Bacillus subtilis. Spo0A is activated by phosphorylation of its regulatory domain by a multicomponent phosphorelay. To define the role of the regulatory domain in the activation of Spo0A, we have characterized four of the sof mutations in vitro. The sof mutations were identified previously as suppressors of the sporulation-negative phenotype resulting from a deletion of the gene for one of the phosphorelay components, spo0F. Like wild-type Spo0A, the transcription stimulation properties of all of the Sof proteins were dependent upon phosphorylation. Sof mutants from two classes were improved substrates for direct phosphorylation by the KinA sensor kinase, providing an explanation for their suppression properties. Two other Sof proteins showed a phosphorylation-dependent enhancement of the stability of the Sof approximately P-RNA polymerase-DNA complex. One of these mutants, Sof114, increased the stability of the Sof114 approximately P-RNAP-DNA complex without increasing its own affinity for the spoIIG promoter. A comparison of the location of the sof mutations with mutations in CheY suggests that phosphorylation of Spo0A results in the exposure of a region in the regulatory domain that interacts with RNA polymerase, thereby contributing to the signal transduction mechanism.

A negative regulator linking chromosome segregation to developmental transcription in Bacillus subtilis.

The SpoOJA and SpoOJB proteins of Bacillus subtilis are similar to the ParA and ParB plasmid-partitioning proteins, respectively, and mutation of spoOJB prevents the expression of stage II genes of sporulation. This phenotype is a consequence of SpoOJA activity in the absence of SpoOJB, and its basis was unknown. In the studies reported here, SpoOJA was found specifically to dissociate transcription initiation complexes formed in vitro by the phosphorylated sporulation transcription factor SpoOA and RNA polymerase with the spollG promoter. This repressor-like activity is likely to be the basis for preventing the onset of differentiation in vivo. SpoOJB is known to neutralize SpoOJA activity in vivo and also to interact with a mitotic-like apparatus responsible for chromosome partitioning. These data suggest that SpoOJA and SpoOJB form a regulatory link between chromosome partition and development gene expression.

The Bacillus subtilis regulator SinR inhibits spoIIG promoter transcription in vitro without displacing RNA polymerase.

Initiation of sporulation in Bacillus subtilis is controlled by several regulators which affect activation by phosphorylation of the key response regulator Spo0A or transcription of Spo0A-P-dependent genes. In vivo overexpression of one of these regulators, sinR , results in suppression of transcription from the Spo0A-P-dependent promoters of spo0A , spoIIA , spoIIE and spoIIG and in vitro SinR binds to the promoters of the spoIIA operon and the spo0A gene. In this study we have demonstrated that in vitro SinR directly repressed Spo0A- P-dependent transcription by B.subtilis RNA polymerase from the spoIIG operon promoter. SinR inhibited transcription prior to formation of heparin-resistant complexes but did not displace RNA polymerase from the spoIIG promoter. DNase I protection studies demonstrated that SinR protected a large region of the spoIIG promoter and induced DNase I hypersensitive sites, particularly around the 0A boxes, at the same positions as those induced by zinc. Since binding of zinc induces bends in the DNA, we concluded that SinR binding also altered the conformation of the spoIIG promoter. We propose that SinR-induced conformational changes in Spo0A-dependent promoters prevent activation of trans-cription by Spo0A-P.

Differences in eucaryotic cell binding of Pseudomonas.

The lungs of cystic fibrosis (CF) patients are frequently chronically colonized by Pseudomonas aeruginosa. Recently there has been an increase in colonization by another pathogen Pseudomonas cepacia, which can cause a rapid decline in clinical condition or death of the patient. The nature of the factor(s) which predispose CF patients to colonization by one or both of these opportunistic pathogens is unknown. It has been suggested that the genetic defect in CF patients results in an increase in the number of epithelial cell receptors available to P. aeruginosa in the lung, thus rendering CF patients more susceptible to bacterial colonization than non-CF individuals. In this study, we have examined adherence of several strains of P. aeruginosa and P. cepacia to a variety of continuous cell lines, as well as primary cultures of CF and non-CF nasal polyp cells. The results suggested that there may be a decrease in the number of receptors available to both strains of Pseudomonas on cells of canine origin compared to human cells. Both strains appear to use pili as the primary adhesin, but there is also evidence that non-pilus adhesins contribute significantly to eucaryotic cell binding. P. cepacia exhibited microcolony formation on all cell types, which is typical of the localized adherence pattern characteristic of the enteropathogenic Escherichia coli. However, we were unable to demonstrate, with either P. cepacia or P. aeruginosa, a significant increase in adherence to CF compared to non-CF nasal polyp cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Fimbriation of Pseudomonas cepacia.

Fimbriae (pili) on the surface of bacteria have been suggested to facilitate adherence to mucosal epithelial surfaces. Three Pseudomonas cepacia cystic fibrosis isolates were screened for their ability to agglutinate erythrocytes (HA), a characteristic of some fimbrial types. One strain, designated PC103, was HA+, while another, PC109, was HA-. A fimbriated (f+) HA+ derivative of PC109 (PC2(13)) was selected by repeated erythrocyte adsorption. The two HA+ strains were shown by transmission electron microscopy to possess fimbriae which averaged 4.8 +/- 1.36 nm in width and 200 to greater than 2,100 nm in length (PCE2(13)) and 3.4 to 11.4 nm in diameter and 280 to 720 nm in length (PC103). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins prepared from PC103, PC109, and PCE2(13) indicated that the putative fimbrial subunit had a mass of 16 kDa. Western blot (immunoblot) analysis of sheared cell supernatants indicated that the 16-kDa subunit from PC103 and PCE2(13) reacted with antibody to the P. aeruginosa PAK pilin subunit. Southern blot analysis of a SalI digest of PC103 DNA showed DNA fragments which hybridized to P. aeruginosa PAK probes containing either the pilin structural gene (pilA) or the pilin accessory genes (pilB, -C, and -D) but not the conserved N-terminal region of pilA. A 15-kb band was common to both hybridizations, indicating that this fragment contains the PC103 fimbrial gene cluster. These results indicated the presence of homology between P. aeruginosa PAK and PC103 fimbriae but also suggested that the P. cepacia fimbriae are not type IV-like. The importance of fimbriae in adherence to A549 cells (type II pneumocytes) was assessed with PC109 (f-) and PCE2(13) (f+). PCE2(13) had an approximately 20-fold-higher level of adherence to A549 cells than PC109. This suggested that fimbriation of P. cepacia is associated with increased adherence in vitro.

Modulation of coronavirus-mediated cell fusion by homeostatic control of cholesterol and fatty acid metabolism.

Cellular susceptibility to fusion mediated by murine coronavirus (mouse hepatitis virus, MHV strain A59) was separated into lipid-dependent and lipid-independent mechanisms with the use of subclones and selected mutants of mouse L-2 fibroblasts. Fusion-resistant L-2 cell mutants had similar cholesterol and fatty acid composition as did their fusion-susceptible parent subclone, and were presumably deficient in a genetically mutable non-lipid, host cell factor (e.g., fusion protein receptor). On the other hand, cellular sensitivity to virus fusion, which is known to be influenced by cell cholesterol content [Daya et al., 1988], was shown further to be modulated by homeostatic alterations in fatty acid metabolism. Cholesterol supplementation of mouse L-2 fibroblasts or of peritoneal macrophages from MHV-susceptible mice elevated susceptibility to viral fusion. Increased fusion susceptibility occurred in cholesterol-supplemented L-2 cells in the absence of any detectable alterations in host cell fatty acid composition, thus demonstrating fusion enhancement by cholesterol alone. L-2 cells cloned by limiting dilution in normal (not cholesterol-supplemented) medium were found to be heterogeneous in cholesterol content. Interestingly, high cholesterol-containing subclones had increased levels of C-18:0, C-18:2, C-20:4, and C-22:6 and markedly reduced levels of C-18:1 fatty acids when compared to low cholesterol-containing subclones. High cholesterol-containing subclones did not show enhanced susceptibility to viral fusion, suggesting that homeostatic alteration of fatty acid metabolism compensated for the increased cholesterol levels and countered the normally fusion-enhancing effect of cholesterol alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Differentiation of acid-pH-dependent and -nondependent entry pathways for mouse hepatitis virus.

Early events of infection of MHV were studied in comparison with those of VSV, which is known to enter cells by an endocytic pathway. Treatment of mouse L-2 fibroblasts with ammonium chloride, chloroquine, or dansylcadaverine inhibited infection of MHV to a much lesser degree than that of VSV, suggesting a relatively minor role for the endocytic pathway and functional endosomes in MHV infection. Endocytosis of MHV and VSV into L-2 cells was assayed by the recovery of infectious (i.e., not uncoated) viruses from homogenates of cells harvested within the first few minutes of infection (and treated with protease to remove surface-bound virus). The results thus suggest that while a small proportion of the MHV inoculum is internalized by endocytosis, productive infection does not depend on functional endocytosis as utilized by VSV. Studies on direct virion-mediated cell fusion showed that MHV can induce fusion at pH 7.4, whereas VSV causes fusion at pH 5.0. Taken together, the above results suggest that MHV enters L-2 cells predominantly by membrane fusion with a non-acidified compartment such as the plasma membrane, endocytic vesicles, or endosomes (prior to their acidification). Results obtained from cell lines which differed in permissiveness to MHV infection suggested that the ability to support MHV infection does not correlate with endocytosis. Rather, nonpermissive cells, such as rat astrocytoma (C-6) and Vero cells, showed higher levels of recoverable internalized MHV than did fully permissive L-2 cells. Cells which are normally nonpermissive to MHV, could be rendered MHV-susceptible by PEG-induced fusion of cell surface-bound virus. Such PEG-mediated susceptibility to MHV infection was insensitive to inhibition by ammonium chloride, supporting the idea that host cell restriction of MHV infection in C-6 and Vero cells may be due to a block in nonendosomal membrane fusion. Thus endocytic internalization of MHV, which clearly occurs in a variety of cells, does not guarantee productive infection.

Mouse fibroblast mutants selected for survival against mouse hepatitis virus infection show increased resistance to infection and virus-induced cell fusion.

We describe here a genetic approach to the analysis of host cell functions involved in determining permissiveness to mouse hepatitis virus (MHV). Using the chemical mutagen, ethyl methane sulfonate (EMS), mouse fibroblast cell mutants were generated which were selected for resistance to cell-killing by MHV. These mutants were then screened for their susceptibility to MHV infection, ability to replicate MHV and relative sensitivity to MHV-induced cell fusion. In contrast to wild type L-2 cells which were acutely and terminally infected by MHV, all five mutants examined replicated MHV in a persistent manner. These mutants showed a reduced susceptibility to MHV infection and an increased resistance to MHV-induced cell fusion. Fusion resistance was specific to that mediated by the MHV E2 protein; mutant as well as wild type L-2 cells were equally sensitive to fusion by polyethylene glycol. The combined effect of reduced infectability and increased fusion resistance was to limit MHV infection to only a small percentage of the total cells in culture, thereby permitting survival of both virus and cells. The observed high rate of generation of the cell mutants suggests that the conversion of a fully MHV-susceptible cell to a semi-resistant one (capable of supporting a persistent infection) is a fairly common event, possibly involving a single mutation.

Mutation of host cell determinants which discriminate between lytic and persistent mouse hepatitis virus infection results in a fusion-resistant phenotype.

The expression of mouse hepatitis virus (MHV) E2-specific mRNA, the E2 polypeptide and its associated cell fusing activity was monitored in various cell types inoculated with a recombinant vaccinia virus, designated vMS containing the E2 gene. The results suggest that host cell permissiveness to MHV infection correlates with cellular susceptibility to membrane fusion mediated by the MHV E2 glycoprotein. In addition, we utilized a genetic approach to the analysis of host cell functions involved in determining permissiveness to MHV. By using the chemical mutagen ethyl methanesulphonate, mouse fibroblast cell mutants were generated and selected for their resistance to cell killing by MHV. When challenged with MHV, all five mutants examined gave rise to persistent infections, in contrast to wild-type L-2 cells which were rapidly killed by the virus. The results provide genetic evidence in support of a previous correlation proposed between MHV permissiveness and two host determinants, namely susceptibility to MHV infection and to MHV-mediated cell fusion. Fusion resistance was specific to fusion mediated by the MHV E2 glycoprotein as shown in contact fusion assays between uninfected cells and cells infected either with MHV or with an E2-expressing recombinant vaccinia virus. In contrast, mutant cells were not resistant to fusion after treatment with polyethylene glycol. The observed high rate of generation of these mutants suggests that the conversion of a fully MHV-susceptible cell to a semi-resistant one is a fairly common event, possibly involving a single mutation. In this case, resistance to MHV infection and to E2-mediated membrane fusion may depend on a common host function. This result provides prospects for the precise genetic and biochemical characterization of the steps involved in host cell permissiveness to MHV infection.

Cholesterol enhances mouse hepatitis virus-mediated cell fusion.

Mouse hepatitis virus (MHV) infection of the L-2 subline of mouse fibroblasts results in acute infection characterized by extensive cell fusion. In contrast, infection of the LM-K subline leads to virus persistence with reduced cell fusion. We undertook studies designed to elucidate the role of host cell membrane lipid composition and the cytoskeleton in modulating the fusion process and the resultant effect(s) on virus persistence. MHV-induced cell fusion proceeded normally in cells treated with cytoskeleton-disrupting drugs, cytochalasin B and colchicine. Modification of cell membrane fatty acid composition by supplementation of LM-K cells with arachidonic (C-20:4) or palmitic (C-16:0) acids had little effect on the extent of MHV-induced cell fusion or on virus replication. However, supplementation of both cell types with cholesterol (resulting in increased membrane cholesterol/fatty acid ratio) resulted in marked enhancement of virus-mediated cell fusion. The increase in cell membrane cholesterol did not enhance internalization of MHV suggesting that cholesterol primarily modulates a later event. This suggestion was confirmed by demonstrating cholesterol-enhancement of fusion in a contact fusion assay. Cholesterol-supplemented L-2 cells were less productive for virus replication than unsupplemented cells, in agreement with our previous observations that MHV replication is compromised by extensive cytopathic effect. Although cholesterol-supplemented LM-K cells showed increased susceptibility to MHV-mediated cell fusion, the extent of such susceptibility did not approach that observed in L-2 cells. Also, the property of LM-K cells to support MHV persistence was not abolished by cholesterol supplementation. Thus membrane fusion resistance and MHV persistence are modulated but not alleviated by cell membrane cholesterol content.