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.     

Characterization of a Pseudomonas putida rough variant evolved in a mixed-species biofilm with Acinetobacter sp. strain C6

Genetic differentiation by natural selection is readily observed among microbial populations, but a more comprehensive understanding of evolutionary forces, genetic causes, and resulting phenotypic advantages is not often sought. Recently, a surface population of Pseudomonas putida bacteria was shown to evolve rapidly by natural selection of better-adapted variants in a mixed-species biofilm consortium (S. K. Hansen, P. B. Rainey, J. A. Haagensen, and S. Molin, Nature 445:533-536, 2007). Adaptation was caused by mutations in a wapH homolog (PP4943) involved in core lipopolysaccharide biosynthesis. Here we investigate further the biofilm physiology and the phenotypic characteristics of the selected P. putida rough colony variants. The coexistence of the P. putida population in a mixed-species biofilm with Acinetobacter sp. strain C6 is dependent on the benzoate excreted from Acinetobacter during the catabolism of benzyl alcohol, the sole carbon source. Examination of biofilm development and the dynamics of the wild-type consortium revealed that the biofilm environment became oxygen limited, possibly with low oxygen concentrations around Acinetobacter microcolonies. In contrast to P. putida wild-type cells, which readily dispersed from the mixed-species biofilm in response to oxygen starvation, the rough variant cells displayed a nondispersal phenotype. However, in monospecies biofilms proliferating on benzoate, the rough variant (like the wild-type population) dispersed in response to oxygen starvation. A key factor explaining this conditional, nondispersal phenotype is likely to be the acquired ability of the rough variant to coaggregate specifically with Acinetobacter cells. We further show that the P. putida rough variant displayed enhanced production of a cellulose-like polymer as a consequence of the mutation in wapH. The resulting phenotypic characteristics of the P. putida rough variant explain its enhanced fitness and ability to form tight structural associations with Acinetobacter microcolonies.     

Second Site Mutations Specifically Suppress the Fix(-) Phenotype of Rhizobium Meliloti Ndvf Mutations on Alfalfa: Identification of a Conditional Ndvf-Dependent Mucoid Colony Phenotype

Rhizobium meliloti mutants carrying ndvF insertion or deletion mutations induce nodules on alfalfa which contain very few infected cells and fail to fix N(2) (Fix(-)). We have characterized five independent second site mutations (designated sfx) which completely suppress the Fix(-) phenotype of ndvF mutants on Medicago sativa but not on another R. meliloti host Melilotus alba. Genetic mapping and phenotypic analysis revealed that the suppressor mutations sfx-1, sfx-4 and sfx-5 mapped to a single locus which was distinct from another locus defined by the sfx-2 and sfx-3 mutations. Tn5-mob-mediated conjugal mapping experiments showed that the sfx-1 locus was located clockwise from trp-33 on the R. meliloti chromosome and a detailed cotransduction map of this region was generated. To clone the sfx-1 locus, we prepared a cosmid library from total DNA obtained from an sfx-1, ndvF deletion strain. From this library, a cosmid pTH56, which converted Fix(-) ndvF mutants to Fix(+), was isolated. Southern blot analysis provided direct physical evidence that the insert DNA in plasmid pTH56 was contiguous with the sfx-1 region. On low osmolarity glutamate-yeast extract-mannitol-salts medium (GYM) agar medium, ndvF insertion and deletion mutants were found to have a mucoid colony phenotype, as opposed to the dry colony phenotype of the wild-type strain. This phenotype was shown to be dependent on the exoB and expE genes required for synthesis of exopolysaccharide II in R. meliloti but not to be dependent on genes required exclusively for the synthesis of the succinoglycan or exopolysaccharide I. Transduction of either sfx-1 or sfx-2 or transfer of the cosmid pTH56 into the ndvF mutants restored them to a wild-type dry colony phenotype. The mucoid phenotype is not responsible for the Fix(-) phenotype of ndvF mutants as the Fix(-), ndvF exp double mutants can be complemented to Fix(+) by introducing plasmids which carry only the wild-type ndvF genes.     

The rbmBCDEF gene cluster modulates development of rugose colony morphology and biofilm formation in Vibrio cholerae

Vibrio cholerae, the causative agent of cholera, can undergo phenotypic variation generating rugose and smooth variants. The rugose variant forms corrugated colonies and well-developed biofilms and exhibits increased levels of resistance to several environmental stresses. Many of these phenotypes are mediated in part by increased expression of the vps genes, which are organized into vps-I and vps-II coding regions, separated by an intergenic region. In this study, we generated in-frame deletions of the five genes located in the vps intergenic region, termed rbmB to -F (rugosity and biofilm structure modulators B to F) in the rugose genetic background, and characterized the mutants for rugose colony development and biofilm formation. Deletion of rbmB, which encodes a protein with low sequence similarity to polysaccharide hydrolases, resulted in an increase in colony corrugation and accumulation of exopolysaccharides relative to the rugose variant. RbmC and its homolog Bap1 are predicted to encode proteins with carbohydrate-binding domains. The colonies of the rbmC bap1 double deletion mutant and bap1 single deletion mutant exhibited a decrease in colony corrugation. Furthermore, the rbmC bap1 double deletion mutant was unable to form biofilms at the air-liquid interface after 2 days, while the biofilms formed on solid surfaces detached readily. Although the colony morphology of rbmDEF mutants was similar to that of the rugose variant, their biofilm structure and cell aggregation phenotypes were different than those of the rugose variant. Taken together, these results indicate that vps intergenic region genes encode proteins that are involved in biofilm matrix production and maintenance of biofilm structure and stability.     

Emergence of a laccase-positive variant of Azospirillum lipoferum occurs via a two-step phenotypic switching process

Two variants have been isolated from the wild-type Azospirillum lipoferum strain 4B. The first variant, 4V(I), spontaneously emerged from the wild-type at frequencies in the order of 10(-4) to 10(-3) per cell generation. Compared to the wild-type, the 4V(I) variant gained (production of a carotenoid-like pigment, assimilation of certain carbohydrates) and lost (swimming motility, reduction of triphenyl tetrazolium chloride, acid production from certain sugars) apparently unrelated phenotypic characteristics. Only from the 4V(I) variant, a second atypical stable form, variant 4V(II), which acquired laccase activity and ability to produce melanin, appeared under very specific conditions, namely growth at extremely low oxygen concentrations. Neither of the variants was able to revert to the parental phenotype. The results suggest that atypical non-motile laccase-positive isolates of A. lipoferum that are found in the rice rhizosphere originate from wild-type (motile, laccase-negative) cells via a two-step phenotypic switching event, a non-motile laccase-negative variant being an intermediate phase.     

The role of the N-terminal oligopeptide repeats of the yeast Sup35 prion protein in propagation and transmission of prion variants

The cytoplasmic [PSI+] determinant of Saccharomyces cerevisiae is the prion form of the Sup35 protein. Oligopeptide repeats within the Sup35 N-terminal domain (PrD) presumably are required for the stable [PSI+] inheritance that in turn involves fragmentation of Sup35 polymers by the chaperone Hsp104. The nonsense suppressor [PSI+] phenotype can vary in efficiency probably due to different inheritable Sup35 polymer structures. Here we study the ability of Sup35 mutants with various deletions of the oligopeptide repeats to support [PSI+] propagation. We define the minimal region of the Sup35-PrD necessary to support [PSI+] as amino acids 1-64, which include the first two repeats, although a longer fragment, 1-83, is required to maintain weak [PSI+] variants. Replacement of wild-type Sup35 with deletion mutants decreases the strength of the [PSI+] phenotype. However, with one exception, reintroducing the wild-type Sup35 restores the original phenotype. Thus, the specific prion fold defining the [PSI+] variant can be preserved by the mutant Sup35 protein despite the change of phenotype. Coexpression of wild-type and mutant Sup35 containing three, two, one, or no oligopeptide repeats causes variant-specific [PSI+] elimination. These data suggest that [PSI+] variability is primarily defined by differential folding of the Sup35-PrD oligopeptide-repeat region.     

Conformation preserved in a weak-to-strong or strong-to-weak [PSI+] conversion during transmission to Sup35 prion variants

The cytoplasmic [PSI(+)] element of budding yeast represents the prion conformation of translation release factor Sup35. Much interest lies in understanding how prions are able to generate variation in isogenic strains. Recent observations suggest that a single prion domain, PrD, is able to adopt several conformations that account for prion strains. We report novel PrD variants of Sup35 that convert weak [PSI(+)] to strong [PSI(+)], and vice versa, upon transmission from wild-type Sup35. During the transmission from wild-type Sup35 to variant Sup35s, no conformational changes were detected by proteolytic fingerprinting and the original [PSI(+)] strain was remembered upon return to wild-type Sup35. These findings suggest that during transmission to variant Sup35s, the [PSI(+)] phenotype is variable while the original conformation is remembered. A mechanism of "conformational memory" to remember specific [PSI(+)] conformations during transmission is proposed.     

Phenotypic characteristics of a slow-growing, nongerminating variant of Candida albicans

Some of the phenotypic characteristics of a slow-growing, nongerminating variant of a commonly studied strain of Candida albicans are described. The variant arose as a chance isolate. The rate of occurrence was about 0 X 1% and the reversion rate was about 1 per 10(6) cells. The colony size was typically smaller than that of the parent and the yeast cells tended not to separate from one another so that catenulate strands of cells (pseudohyphae) were formed. Under standard conditions the generation time of the small-colony variant in liquid shake cultures was about twice that of the parental strain. Growth of the variant was suppressed by antimycin A, indicating that the small colony form was not the consequence of a defect in the cytochrome system. The colony size of the variant was not influenced by chlorobenzotriazole, which suggested that adenine metabolism was not involved in the small-colony phenotype. The pseudohyphal growth pattern was not relieved by high concentrations of utilizable carbohydrates, which means the catenulate microscopic appearance of the yeast cells was not simply an exaggeration of the normal growth pattern of isolates of C. albicans but more probably represented the growth of a cell-cycle mutant defective at the cell separation step. The cytoplasmic proteins of the variant and the parent were very similar though some unique peptides were displayed by each.     

Analysis of second-site revertants of a murine coronavirus nucleocapsid protein deletion mutant and construction of nucleocapsid protein mutants by targeted RNA recombination.

The Alb4 mutant of the coronavirus mouse hepatitis virus (MHV) is both temperature sensitive and thermolabile owing to a deletion in the gene encoding its nucleocapsid (N) protein. The deletion removes 29 amino acids that constitute a putative spacer region preceding the carboxyl-terminal domain of the protein. As a step toward understanding the structure and function of the MHV N protein, we isolated multiple independent revertants of Alb4 that totally or partially regained the ability to form large (wild-type-sized) plaques at the nonpermissive temperature. The N proteins of these revertant viruses concomitantly regained the ability to bind to RNA in vitro at a temperature that was restrictive for RNA binding by Alb4 N protein. Sequence analysis of the N genes of the revertants revealed that each contained a single second-site point mutation that compensated for the effects of the deletion. All reverting mutations were clustered within a stretch of 40 amino acids centered some 80 residues on the amino side of the Alb4 deletion, within a domain to which the RNA-binding activity of N had been previously mapped. By means of a targeted RNA recombination method that we have recently developed, two of the reverting mutations were introduced into a wild-type MHV genomic background. The resulting recombinants were stable and showed no gross phenotypic differences from the wild type. A detailed analysis of one, however, revealed that it was at a selective disadvantage with respect to the wild type.     

苏云金芽胞杆菌LM1212菌株和突变株的全基因组序列比较分析

【背景】LM1212菌株是昆虫病原菌苏云金芽胞杆菌(Bacillus thuringiensis,Bt)中的一员,其芽胞和晶体分别产生于芽胞形成细胞和晶体产生细胞中,具有独特的细胞分化表型。与野生株LM1212相比,突变株LM1212-DB芽胞细胞比例明显降低并产生更高比例的晶体产生细胞,这使得LM1212-DB菌株成为研究晶体产生细胞形成机制和提高菌株杀虫活性的绝佳实验材料。【目的】比较LM1212菌株和LM1212-DB菌株的基因组差异,以便于揭示导致这两个菌株表型差异的原因。【方法】利用单分子测序技术(single molecular real-time,SMRT)和Pacbio RS II测序平台对两个菌株进行全基因组测序,对染色体和质粒、双组分信号系统和插入序列等进行差异分析,并构建表型特性相关基因的系统发育树。【结果】基因组分析发现,LM1212和LM1212-DB菌株均含有丰富的插入序列和双组分信号系统,暗示两个菌株极易发生基因重排且具有较强的环境适应性。与LM1212菌株相比,突变株LM1212-DB中发生了染色体和质粒片段缺失、质粒重排、质粒拷贝数变异。进一步分析缺失基因的功能发现,一些环境胁迫响应基因(如sigB)和芽胞形成相关基因(如abrB)等缺失;通过分析质粒拷贝数变异发现,具有增加晶体细胞比例功能的转录因子CpcR所在质粒的拷贝数增加1个,同时对CpcR的进化分析发现,与其亲缘关系最近的基因的从属菌株也产生与LM1212菌株相似的细胞分化表型。这些重要功能基因的缺失和拷贝数变异可能是导致两个菌株表型差异的原因。此外,突变株LM1212-DB缺失I型限制-修饰系统,这使得突变株LM1212-DB与野生菌株LM1212相比具有更好的外源DNA兼容性。【结论】突变株LM1212-DB染色体和质粒的结构变异可能是导致与野生株LM1212表型差异的潜在原因,这将为研究LM1212菌株的晶体细胞分化机制提供指导方向。     

Phenotypic variability in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strains Sp7 and Sp245: Association with dormancy and characteristics of the variants

The state of metabolic dormancy in diazotrophic bacteria Azospirillum brasilense Sp7 (non-endophytic strain) and Sp245 (endophytic strain) was found to be associated with phenotypic variability. The latter manifested itself in the extension of the spectrum of A. brasilense phenotypic variants resulting from plating of cyst-like resting cells (CRC) on solid media and was more pronounced in strain Sp7. The major colony’s morphological variants of strain Sp7 were (1) the dominant S type; (2) the highly pigmented Pg type; (3) the R type; (4) the Sm type, forming small colonies; and (5) the Sg type, forming segmented colonies. In addition to their colony morphology, the variants differed in the phenotype stability during transfers on the standard solid medium and in their motility in semisolid agar. The occurrence frequency of the phenotypic variants depended on the conditions and duration of incubation (storage) of the CRC of strain Sp7, as well as on heat treatment (at 55 and 60°C for 10 min) of the cells prior to inoculation. The maximum frequency of S → Pg transitions (up to 74%) was observed during the germination of CRC stored in a spent culture medium at −20°C for 4 months; the maximum frequency (up to 100%) of S → Sm transitions was observed after inoculation of the CRC subjected to heat treatment. The Pg variants were the most stable, whereas other types reverted rapidly to the S or Pg variant. The S variant grown in semisolid agar exhibited the mixed type of motility (Swa⁺Gri⁺, swarming and migration in the form of microcolonies); the Pg and Sg variants showed the Swa⁺Gri⁻ (swarming) phenotype and the Sm variant was nonmotile (Swa⁻Gri⁻ phenotype). The spectrum of phenotypic variants of the endophytic strain Sp245 was narrower than that of strain Sp7 and was represented by S, Sm, and M (mucoid) variants that differed in the patterns of cell motility: the dominant S type displayed the swarming pattern (Swa⁺Gri⁻), the mucoid M type showed the mixed type (Swa⁺Gri⁺) of motility, and the Sm variant was nonmotile. The differences between the nonendophytic strain Sp7 and the endophytic strain Sp245 in their capacity for phenotypic dissociation and cell motility in semisolid media may reflect their ability to adapt to changing ambient conditions and specificity of plant-microbial interactions.     

Stability and activity improvement of cephalosporin esterase EstB from Burkholderia gladioli by directed evolution and structural interpretation of muteins

Esterase EstB from Burkholderia gladioli, which belongs to a family of esterases related to beta-lactamases and DD-peptidases was evolved for increased stability and simultaneously maintaining high cephalosporin C deacetylation activity. Random mutagenesis PCR was used to generate up to 5 aa substitutions per gene. A newly designed colony filter-screening assay, which was based on pH change after deacetylation of cephalosporin C in presence of DMF was established. In a first evolution round employing random mutagenesis, which included about 10(6) mutants, a set of interesting mutants was isolated. Distinct mutations identified as significant for stability were combined by a rational recombination step and the resulting recombinant was further evolved by an additional random mutagenesis round. After screening an additional 10(5) clones, it was possible to isolate a variant of EstB having more than 100-fold better activity in reactions containing 35% DMF. This mutant also showed a high increase in temperature stability (T(m) was raised by 13 degrees C) and retained high activity towards cephalosporin C under standard assay conditions. The molecular effects of mutations found in random mutants are discussed in view of the three-dimensional structure of wild-type EstB.     

Molecular nature of spontaneous modifications in gacS which cause colony phase variation in Pseudomonas sp. strain PCL1171

Pseudomonas sp. strain PCL1171 displays colony phase variation between opaque phase I and translucent phase II colonies, thereby regulating the production of secondary metabolites and exoenzymes. Complementation and sequence analysis of 26 phase II mutants and of 13 wild-type phase II sectors growing out of phase I colonies showed that in all these cases the phase II phenotype is caused by spontaneous mutations in gacA or/and gacS. Mutation of gac reduced both the length of the lag phase and the generation time. Isolation and sequencing of the gacS genes from the phase II bacteria revealed one insertion as well as several random point mutations, deletions, and DNA rearrangements. Most phase II colonies reverted with a high frequency, resulting in wild-type gacA and gacS genes and a phase I phenotype. Some phase II bacteria retained the phase II phenotype but changed genotypically as a result of (re)introduction of mutations in either gacA or gacS. The reversion of gacA or gacS to the wild type was not affected by mutation of recA and recB. We conclude that in Pseudomonas sp. strain PCL1171, mutations in gacA and gacS are the basis for phase variation from phase I to phase II colonies and that, since these mutations are efficiently removed, mutations in gac result in dynamic switches between the "wild-type" population and the subpopulations harboring spontaneous mutations in gacA and or gacS, thereby enabling both populations to be maintained.     

Phase variation, capsular polysaccharide, pilus and flagella contribute to uptake of Vibrio vulnificus by the Eastern oyster (Crassostrea virginica)

Vibrio vulnificus infections are associated with raw oyster consumption, and disease reservoirs are determined by the ability of this bacterium to infect and persist in oysters. Surface structures, such as capsular polysaccharide (CPS), pili and flagella, function as virulence factors in mouse infection models. Furthermore, virulence is related to phase variation in colony morphology, which reflects CPS expression and includes opaque (encapsulated, virulent), translucent (reduced encapsulation, avirulent) and rugose (wrinkled, biofilm-enhanced) colony types. The role of these factors in environmental survival is unknown; therefore, mutational analysis and phase variation of V. vulnificus were examined in an oyster infection model. Oysters ( Crassostrea virginica ) were pre-treated with tetracycline to reduce background bacteria and subsequently inoculated via filter feeding with 10⁶ colony-forming units (cfu) ml⁻¹ of V. vulnificus wild-type strains and phase variants, as well as strains with deletion mutations in genes related to CPS (Δ wza ), pili (Δ pilA ), flagella (Δ flaCDE/ Δ flaFBA ) and motility (Δ motAB ). All mutants were significantly reduced in their dissemination to oyster haemolymph as compared with wild type; however, recovery of mutants from gills and intestinal tissue was generally similar to wild type. Translucent and rugose inocula showed induction of high-frequency phase variation to the opaque encapsulated phenotype (100% and 72% respectively) during oyster infections that did not occur in strains recovered from seawater. Thus, multiple bacterial factors determine uptake of V. vulnificus in oysters, and phase variation during oyster infection is a likely mechanism for environmental survival and for induction of the more virulent phenotype.     

FvVE1 regulates filamentous growth, the ratio of microconidia to macroconidia and cell wall formation in Fusarium verticillioides

The velvet gene, veA, co-ordinates asexual and sexual development in the homothallic fungal species Aspergillus nidulans. Studies in Aspergillus parasiticus and Aspergillus fumigatus demonstrated that veA also regulates morphological differentiation in these species. Whether veA has the same role in morphogenesis in other fungal genera has not been investigated. In this work, we studied the role of the veA homologue, FvVE1, in the heterothallic fungus Fusarium verticillioides. Deletion of FvVE1 suppressed aerial hyphal growth and reduced colony surface hydrophobicity on solid media. In submerged cultures, FvVE1 deletion caused alterations in hyphal polarity, marked activation of conidiation and yeast-like growth. The latter was promoted by shaking to increase aeration of cultures. In addition, FvVE1 deletion markedly increased the ratio of macroconidia to microconidia. Supplementation of osmotic stabilizers restored the wild-type phenotype to deletion mutants, suggesting phenotypic alterations caused by FvVE1 deletion are related to cell wall defects. This is consistent with the hypersensitivity of FvVE1 deletion mutants to SDS and with the significant reduction in the mannoprotein content of mutants compared with the wild-type strain. However, no dramatic cell wall alterations were observed when mutants were examined by transmission electron microscopy. Our data strongly suggest that FvVE1 is important for cell wall integrity, cell surface hydrophobicity, hyphal polarity and conidiation pattern.     

Phase and antigenic variation mediated by genome modifications

Phase and antigenic variation is used by several bacterial species to generate intra-population diversity that increases bacterial fitness and is important in niche adaptation, or to escape host defences. By this adaptive process, bacteria undergo frequent and usually reversible phenotypic changes resulting from genetic or epigenetic alterations at specific genetic loci. Phase variation or phenotypic switch allows the expression of a given phenotype to be switched ON or OFF. Antigenic variation refers to the expression of a number of alternative forms of an antigen on the cell surface, and at a molecular level, shares common features with phase variation mechanisms. This review will focus on phase and antigenic variation mechanisms implying genome modifications, with an emphasis on the diversity of phenotypes regulated by these mechanisms, and the ecological relevance of variant appearance within a given population.