Cloning of wild-type Pseudomonas solanacearum phcA, a gene that when mutated alters expression of multiple traits that contribute to virulence.

Pseudomonas solanacearum undergoes a spontaneous mutation that pleiotropically reduces extracellular polysaccharide (EPS) production, endoglucanase activity, and virulence and increases motility. We refer to the process that coordinately affects these traits as phenotype conversion (PC) and the resulting mutants as PC types. Previous research with the wild-type strain AW1 suggested that inactivation of a single locus could mimic phenotype conversion (T. P. Denny, F. W. Makini, and S. M. Brumbley, Mol. Plant-Microbe Interact. 1:215-223, 1988). Additional Tn5 mutagenesis of AW1 generated three more mutants (AW1-81, AW1-82, and AW1-84) that were indistinguishable from the PC type and one slightly leaky mutant (AW1-87); all four had single insertions in the same 4.0-kilobase (kb) EcoRI fragment that were responsible for the PC-like phenotype. Another insertion mutant, AW1-83, which lacks an insertion in this 4.0-kb fragment, resembled the PC type except that it was reversibly induced to produce wild-type levels of EPS when cultured adjacent to AW1. The wild-type region containing the gene that controls traits affected by phenotype conversion in AW1, designated phcA, was cloned on a 2.2-kb DNA fragment that restored all the phcA::Tn5 mutants and 11 independent spontaneous PC-type derivatives of AW1 to wild-type status. Homology with the phcA region was found in diverse wild-type strains of P. solanacearum, although restriction fragment length polymorphisms were seen. No major DNA alterations were observed in the phcA homologous region of PC types from strain AW1 or 82N. PC types from 7 of 11 conjugal strains of P. solanacearum were restored to EPS+ by phcA from AW1; however, only some PC types of strain K60 were restored, whereas others were not. We believe that a functional phcA gene is required to maintain the wild-type phenotype in P. solanacearum, and for most strains phenotype conversion results from a loss of phcA gene expression or the function of its gene product.     

Isolation and characterization of cell wall-defective variants of Bacillus subtilis and Bacillus licheniformis

Quantitative mass conversion of intact bacterial cells of Bacillus subtilis and B. licheniformis to L-phase variants has been effected after lysozyme treatment. After subculture of the unstable L-phase variants for several months in the presence of methicillin, stable L-phase variants were obtained which grew in the absence of the antibiotic and were then unable to revert to the classical bacterial phase under conditions which gave rise to mass reversion of the protoplasts and unstable L-variants. These stable L-phase variants, which retained many of the physiological properties of the bacterium from which they were derived, were capable of growing exponentially and multiplying in liquid medium. Their morphology and apparent modes of reproduction were consistent with that described for other L-phase variants. The morphological events, as monitored by the electron microscope, of the reversion to the intact bacterial phase of an unstable L-phase variant of B. licheniformis are described.     

Identification and characterization of a locus which regulates multiple functions in Pseudomonas tolaasii, the cause of brown blotch disease of Agaricus bisporus.

Pseudomonas tolaasii, the causal agent of brown blotch disease of Agaricus bisporus, spontaneously gives rise to morphologically distinct stable sectors, referred to as the phenotypic variant form, at the margins of the wild-type colonies. The phenotypic variant form is nonpathogenic and differs from the wild type in a range of biochemical and physiological characteristics. A genomic cosmid clone (pSISG29) from a wild-type P. tolaasii library was shown to be capable of restoring a range of characteristics of the phenotypic variant to those of the wild-type form, when present in trans. Subcloning and saturation mutagenesis analysis with Tn5lacZ localized a 3.0-kb region from pSISG29, designated the pheN locus, required for complementation of the phenotypic variant to the wild-type form. Marker exchange of the Tn5lacZ-mutagenized copy of the pheN locus into the wild-type strain demonstrated that a functional copy of the pheN gene is required to maintain the wild-type pathogenic phenotype and that loss of the pheN gene or its function results in conversion of the wild-type form to the phenotypic variant form. The pheN locus contained a 2,727-bp open reading frame encoding an 83-kDa protein. The predicted amino acid sequence of the PheN protein showed homology to the sensor and regulator domains of the conserved family of two component bacterial sensor regulator proteins. Southern hybridization analysis of pheN genes from the wild type and the phenotypic variant form revealed that DNA rearrangement occurs within the pheN locus during phenotypic variation. Analysis of pheN expression with a pheN::lacZ fusion demonstrated that expression is regulated by environmental factors. These results are related to a model for control for phenotypic variation in P. tolaasii.     

In vivo formation of allosteric aspartate transcarbamoylase containing circularly permuted catalytic polypeptide chains: implications for protein folding and assembly.

Because the N- and C-terminal amino acids of the catalytic (c) polypeptide chains of Escherichia coli aspartate transcarbamoylase (ATCase) are in close proximity to each other, it has been possible to form in vivo five different active ATCase variants in which the terminal regions of the wild-type c chains are linked in a continuous polypeptide chain and new termini are introduced elsewhere in either of the two structural domains of the c chain. These circularly permuted (cp) chains were produced by constructing tandem pyrB genes, which encode the c chain of ATCase, followed by application of PCR. Chains expressed in this way assemble efficiently in vivo to form active, stable ATCase variants. Three such variants have been purified and shown to have the kinetic and physical properties characteristic of wild-type ATCase composed of two catalytic (C) trimers and three regulatory (R) dimers. The values of Vmax for cpATCase122, cpATCase222, and cpATCase281 ranged from 16-21 mumol carbamoylaspartate per microgram per h, compared with 15 for wild-type ATCase, and the values for K0.5 for the variants were 4-17 mM aspartate, whereas wild-type ATCase exhibited a value of 6 mM. Hill coefficients for the three variants varied from 1.8 to 2.1, compared with 1.4 for the wild-type enzyme. As observed with wild-type ATCase, ATP activated the variants containing the circularly permuted chains, as shown by the lowering of K0.5 for aspartate and a decrease in the Hill coefficient (nH). In contrast, CTP caused both an increase in K0.5 and nH for the variants, just as observed with wild-type ATCase. Thus, the enzyme containing the permuted chains with widely diverse N- and C-termini exhibited the homotropic and heterotropic effects characteristic of wild-type ATCase. The decrease in the sedimentation coefficient of the variants caused by the binding of the bisubstrate ligand N-(phosphonacetyl)-L-aspartate (PALA) was also virtually identical to that obtained with wild-type ATCase, thereby indicating that these altered ATCase molecules undergo the analogous ligand-promoted allosteric transition from the taut (T) state to the relaxed (R) conformation. These ATCase molecules with new N- and C-termini widely dispersed throughout the c chains are valuable models for studying in vivo and in vitro folding of polypeptide chains.     

Tumor suppressor p53: analysis of wild-type and mutant p53 complexes.

It has been suggested that the dominant effect of mutant p53 on tumor progression may reflect the mutant protein binding to wild-type p53, with inactivation of suppressor function. To date, evidence for wild-type/mutant p53 complexes involves p53 from different species. To investigate wild-type/mutant p53 complexes in relation to natural tumor progression, we sought to identify intraspecific complexes, using murine p53. The mutant phenotype p53-246(0) was used because this phenotype is immunologically distinct from wild-type p53-246+ and thus permits immunological analysis for wild-type/mutant p53 complexes. The p53 proteins were derived from genetically defined p53 cDNAs expressed in vitro and also from phenotypic variants of p53 expressed in vivo. We found that the mutant p53 phenotype was able to form a complex with the wild type when the two p53 variants were cotranslated. When mixed in their native states (after translation), the wild-type and mutant p53 proteins did not exhibit any binding affinity for each other in vitro. Under identical conditions, complexes of wild-type human and murine p53 proteins were formed. For murine p53, both the wild-type and mutant p53 proteins formed high-molecular-weight complexes when translated in vitro. This oligomerization appeared to involve the carboxyl terminus, since truncated p53 (amino acids 1 to 343) did not form complexes. We suggest that the ability of the mutant p53 phenotype to complex with wild type during cotranslation may contribute to the transforming function of activated mutants of p53 in vivo.     

Phenotypic variation of Pseudomonas putida and P. tolaasii affects the chemotactic response to Agaricus bisporus mycelial exudate.

The chemotactic response of wild-type Pseudomonas putida and P. tolaasii, and a phenotypic variant of each species, to Agaricus bisporus mycelial exudate was examined. Both P. putida, the bacterium responsible for initiating basidiome development of A. bisporus, and P. tolaasii, the causal organism of bacterial blotch disease of the mushroom, displayed a positive chemotactic response to Casamino acids and to A. bisporus mycelial exudate. The response was both dose- and time-dependent and marked differences were observed between the response time of the wild-type strains and their phenotypic variants. Phenotypic variants responded rapidly to both attractants and reached a maximum response after 10-20 min, whereas the wild-types took 45-60 min. The differences are partly explained by the more rapid swimming speed of the phenotypic variants. Both variants responded maximally to similar concentrations of Casamino acids and mycelial exudates. Investigations into the nature of the attractants contained in the mycelial exudate indicated that they are predominantly small (Mr less than 2000) thermostable compounds. Sugars present in the exudate did not elicit a chemotactic response in any isolate, but a mixture of 14 amino acids detected in the exudate accounted for between 50 and 75% of the chemotactic response of the fungal exudate.     

The L-Phase of Erwinia carotovora var. atroseptica and its Possible Association with Plant Tissue

S ummary . The growth and recognition of the L-phase of Erwinia carotovora var. atroseptica in vitro and in vivo is described. Evidence is offered for its existence in nature. It is postulated that the bacterium may be present in the potato plant in such a form, closely associated with the plant tissue cells, and as such may be the means of transmitting the disease from one plant to another without the disease being apparent. Under certain circumstances, as yet undetermined, changes in the environment may lead to the reversion of the L-phase to the bacterial phase with the consequent development of the characteristic disease syndrome.     

Complementation analysis in Fanconi anemia: assignment of the reference FA-H patient to group A

Fanconi anemia (FA) is an autosomal recessive disorder with diverse clinical symptoms and extensive genetic heterogeneity. Of eight FA genes that have been implicated on the basis of complementation studies, four have been identified and two have been mapped to different loci; the status of the genes supposed to be defective in groups B and H is uncertain. Here we present evidence indicating that the patient who has been the sole representative of the eighth complementation group (FA-H) in fact belongs to group FA-A. Previous exclusion from group A was apparently based on phenotypic reversion to wild-type rather than on genuine complementation in fusion hybrids. To avoid the pitfall of reversion, future assignment of patients with FA to new complementation groups should conform with more-stringent criteria. A new group should be based on at least two patients with FA whose cell lines are excluded from all known groups and that fail to complement each other in fusion hybrids, or, if only one such cell line were available, on a new complementing gene that carries pathogenic mutations in this cell line. On the basis of these criteria, the current number of complementation groups in FA is seven.     

The sequence-dependent unfolding pathway plays a critical role in the amyloidogenicity of transthyretin

Human transthyretin (TTR) is an amyloidogenic protein whose aggregation is associated with several types of amyloid diseases. The following mechanism of TTR amyloid formation has been proposed. TTR tetramer at first dissociates into native monomers, which is the rate-limiting step in fibril formation. The monomeric species then partially unfold to form amyloidogenic intermediates that subsequently undergo a downhill self-assembly process. The amyloid deposit can be facilitated by disease-associated point mutations. However, only subtle structural differences were observed between the crystal structures of the wild type and the disease-associated variants. To investigate how single-point mutations influence the effective energy landscapes of TTR monomers, molecular dynamics (MD) simulations were performed on wild-type TTR and two pathogenic variants. Principal coordinate analysis on MD-generated ensembles has revealed multiple unfolding pathways for each protein. Amyloidogenic intermediates with the dislocated C strand-loop-D strand motif were observed only on the unfolding pathways of V30M and L55P variants and not for wild-type TTR. Our study suggests that the sequence-dependent unfolding pathway plays a crucial role in the amyloidogenicity of TTR. Analyses of side chain concerted motions indicate that pathogenic mutations on "edge strands" disrupt the delicate side chain correlated motions, which in turn may alter the sequence of unfolding events.     

Polymorphism at residue 129 modulates the conformational conversion of the D178N variant of human prion protein 90-231

One of the arguments in favor of the protein-only hypothesis of transmissible spongiform encephalopathies is the link between inherited prion diseases and specific mutations in the PRNP gene. One such mutation (Asp178 --> Asn) is associated with two distinct disorders: fatal familial insomnia or familial Creutzfeldt-Jakob disease, depending upon the presence of Met or Val at position 129, respectively. In this study, we have characterized the biophysical properties of recombinant human prion proteins (huPrP90-231) corresponding to the polymorphic variants D178N/M129 and D178N/V129. In comparison to the wild-type protein, both polymorphic forms of D178N huPrP show a greatly increased propensity for a conversion to beta-sheet-rich oligomers (at acidic pH) and thioflavine T-positive amyloid fibrils (at neutral pH). Importantly, the conversion propensity for the D178N variant is strongly dependent upon the M/V polymorphism at position 129, whereas under identical experimental conditions, no such dependence is observed for the wild-type protein. Amyloid fibrils formed by wild-type huPrP90-231 and the D178N variant are characterized by different secondary structures, and these structures are further modulated by residue 129 polymorphism. Although on the basis of only in vitro data, this study strongly suggests that polymorphism-dependent phenotypic variability of familial prion diseases may be linked to differences in biophysical properties of prion protein variants.     

小菌落突变株：一种利于持续复发性感染的细菌致病形式

小菌落突变株是一种具有独特表型及致病特征且生长缓慢的细菌亚群。在表型上,小菌落突变株表现为生长率低、菌落形态不规则及生化特性异常,这使得临床微生物学家在鉴定时遇到了挑战。在临床上,小菌落突变株比相应的野生株更能持续存在于哺乳动物细胞中,且对抗生素更不敏感。当宿主细胞形成应急的保护性环境时,小菌落突变株会引发隐性或复发性感染。这篇综述涵盖了小菌落突变株相关表型、遗传及临床上的特征,重点描述目前研究最多的葡萄球菌。     

Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections

Small colony variants constitute a slow-growing subpopulation of bacteria with distinctive phenotypic and pathogenic traits. Phenotypically, small colony variants have a slow growth rate, atypical colony morphology and unusual biochemical characteristics, making them a challenge for clinical microbiologists to identify. Clinically, small colony variants are better able to persist in mammalian cells and are less susceptible to antibiotics than their wild-type counterparts, and can cause latent or recurrent infections on emergence from the protective environment of the host cell. This Review covers the phenotypic, genetic and clinical picture associated with small colony variants, with an emphasis on staphylococci, for which the greatest amount of information is available.     

Visualization of harpin secretion in planta during infection of apple seedlings by Erwinia amylovora

Erwinia amylovora is a Gram-negative pathogenic bacterium that infects pear and apple trees as well as other plants from the Rosaceae family. E. amylovora pathogenicity is dependent on a functional Hrp type III secretion system. Harpin, a protein playing a major role in virulence, has been shown to be exported in vitro via the type III secretion apparatus. The data presented here focus on harpin detection in planta after infection of apple seedlings with the wild-type strain CFPB 1430. Using a specific harpin antiserum, harpin was not detected inside the host plant cells, but was found associated with the bacteria and secreted. The extracellular localization of harpin is in agreement with the physiological effects induced by purified harpin when applied as an exogenous elicitor. Harpin was not found associated with the host plant cell wall, a result that weakens its postulated role in cell wall loosening. A differential labelling was observed at the bacterial level: for some bacteria, harpin was exclusively cytoplasmic, whereas in others, it appeared as small patches over the bacterial outer membrane or associated with extracellular linear structures. All the bacteria present within the same area were similarly labelled, suggesting co-ordination in the secretion process. All observations suggest that harpin is synthesized in the bacterial cytoplasm and that secretion occurs from this cytoplasmic pool upon sensing of a plant or bacterial signal.     

Induction of mutation in vitro in phage phi X174 am3 by N4-aminodeoxycytidine triphosphate

When phi X174 am3-phage-infected E. coli is treated with N4-aminocytidine, reversion of the phage to the wild type is efficiently induced. The mechanism of this reversion is considered to consist of metabolic conversion of N4-aminocytidine into its deoxynucleoside 5'-triphosphate followed by incorporation of the nucleotide into the replicating phage DNA, thereby causing AT-to-GC transition at the am3 locus. The second half of this mechanism has now been experimentally proved, using an in vitro mutagenesis system. Thus, by nick-translation, N4-aminodeoxycytidine 5'-triphosphate was incorporated into the replicative form of phi X174 am3 DNA, and the DNA was used to transfect CA++-treated E. coli HF4714 (sup+). The reversion frequency of the phage produced was up to one-order of magnitude greater than that of the control in which the nick-translation had been done without the addition of N4-aminodeoxycytidine triphosphate. This nucleotide analog may be useful as a reagent for in vitro site-directed mutagenesis.     

Control of the Ralstonia solanacearum Type III secretion system (Hrp) genes by the global virulence regulator PhcA

Expression of several virulence factors in the plant pathogen bacterium Ralstonia solanacearum is controlled by a complex regulatory network, at the center of which is PhcA. We provide genetic evidence that PhcA also represses the expression of hrp genes that code for the Type III protein secretion system, a major pathogenicity determinant in this bacterium. The repression of hrp genes in complete medium is relieved in a phcA mutant and two distinct signals, a quorum-sensing signal and complex nitrogen sources, appear to trigger this PhcA-dependent repression. This control of hrp gene expression by PhcA is realized at the level of the HrpG regulatory protein.     

Flagellin is not a major defense elicitor in Ralstonia solanacearum cells or extracts applied to Arabidopsis thaliana

The phytopathogenic bacterium Ralstonia solanacearum requires motility for full virulence, and its flagellin is a candidate pathogen-associated molecular pattern that may elicit plant defenses. Boiled extracts from R. solanacearum contained a strong elicitor of defense-associated responses. However, R. solanacearum flagellin is not this elicitor, because extracts from wild-type bacteria and fliC or flhDC mutants defective in flagellin production all elicited similar plant responses. Equally important, live R. solanacearum caused similar disease on Arabidopsis ecotype Col-0, regardless of the presence of flagellin in the bacterium or the FLS2-mediated flagellin recognition system in the plant. Unlike the previously studied flg22 flagellin peptide, a peptide based on the corresponding conserved N-terminal segment of R. solanacearum, flagellin did not elicit any response from Arabidopsis seedlings. Thus recognition of flagellin plays no readily apparent role in this pathosystem. Flagellin also was not the primary elicitor of responses in tobacco. The primary eliciting activity in boiled R. solanacearum extracts applied to Arabidopsis was attributable to one or more proteins other than flagellin, including species purifying at approximately 5 to 10 kDa and also at larger molecular masses, possibly due to aggregation. Production of this eliciting activity did not require hrpB (positive regulator of type III secretion), pehR (positive regulator of polygalacturonase production and motility), gspM (general secretion pathway), or phcA (LysR-type global virulence regulator). Wild-type R. solanacearum was virulent on Arabidopsis despite the presence of this elicitor in pathogen extracts.     

Increased mutation rates in doubly viral transformed Chinese hamster cells.

An untransformed parental Chinese hamster cell line, one polyoma transformed clone, and two clones containing both SV40 and polyoma DNA were tested for the appearance of variants resistant to 6-thioguanine or oubain. The frequency of such variants was found to be highest in doubly transformed cells. The mutation rate at these loci was correlated with the level of transformation. The mutants observed were stable and had low reversion frequencies with no gross cytogenetic changes. These results establish a quantitative basis for the evaluation of phenotypic variability observed in transformed cell populations.     

Identification of a key amino acid of LuxS involved in AI-2 production in Campylobacter jejuni

Autoinducer-2 (AI-2) mediated quorum sensing has been associated with the expression of virulence factors in a number of pathogenic organisms and has been demonstrated to play a role in motility and cytolethal distending toxin (cdt) production in Campylobacter jejuni. We have initiated the work to determine the molecular basis of AI-2 synthesis and the biological functions of quorum sensing in C. jejuni. In this work, two naturally occurring variants of C. jejuni 81116 were identified, one producing high-levels of AI-2 while the other is defective in AI-2 synthesis. Sequence analysis revealed a G92D mutation in the luxS gene of the defective variant. Complementation of the AI-2(-) variant with a plasmid encoded copy of the wild-type luxS gene or reversion of the G92D mutation by site-directed mutagenesis fully restored AI-2 production by the variant. These results indicate that the G92D mutation alone is responsible for the loss of AI-2 activity in C. jejuni. Kinetic analyses showed that the G92D LuxS has a ～100-fold reduced catalytic activity relative to the wild-type enzyme. Findings from this study identify a previously undescribed amino acid that is essential for AI-2 production by LuxS and provide a unique isogenic pair of naturally occurring variants for us to dissect the functions of AI-2 mediated quorum sensing in Campylobacter.