GAC Mutation Research Articles

Uncovering the GacS-mediated role in evolutionary progression through trajectory reconstruction in Pseudomonas aeruginosa.

The genetic diversities of subpopulations drive the evolution of pathogens and affect their ability to infect hosts and cause diseases. However, most studies to date have focused on the identification and characterization of adaptive mutations in single colonies, which do not accurately reflect the phenotypes of an entire population. Here, to identify the composition of variant subpopulations within a pathogen population, we developed a streamlined approach that combines high-throughput sequencing of the entire population cells with genotyping of single colonies. Using this method, we reconstructed a detailed quorum-sensing (QS) evolutionary trajectory in Pseudomonas aeruginosa. Our results revealed a new adaptive mutation in the gacS gene, which codes for a histidine kinase sensor of a two-component system (TCS), during QS evolution. This mutation reduced QS activity, allowing the variant to sweep throughout the whole population, while still being vulnerable to invasion by the emerging QS master regulator LasR-null mutants. By tracking the evolutionary trajectory, we found that mutations in gacS facilitated QS-rewiring in the LasR-null mutant. This rapid QS revertant caused by inactive GacS was found to be associated with the promotion of ribosome biogenesis and accompanied by a trade-off of reduced bacterial virulence on host cells. In conclusion, our findings highlight the crucial role of the global regulator GacS in modulating the progression of QS evolution and the virulence of the pathogen population.

Two mutations in the transforming growth factor beta-induced gene associated with familial Lattice corneal dystrophy.

To report a phenotypic variant pedigree of lattice corneal dystrophy (LCD) associated with two mutations, R124C and A546D, in the transforming growth factor beta-induced gene (TGFBI). A detailed ocular examination was taken for all participants of a LCD family. Peripheral blood leukocytes from each participant were extracted to obtain the DNA. Polymerase chain reaction (PCR) of all seventeen exons of TGFBI gene was performed. The products were sequenced and analyzed. Histological examination was carried out after a penetrating keratoplasty from the right eye of proband. Genetic analysis showed that the proband and all 6 affected individuals harbored both a heterozygous CGC to TGC mutation at codon 124 and a heterozygous GCC to GAC mutation at codon 546 of TGFBI. None of the 100 control subjects and unaffected family members was positive for these two mutations. Ocular examination displayed multiple refractile lattice-like opacities in anterior stroma of the central cornea and small granular deposits in the peripheral cornea. The deposits were stained positively with Congo red indicating be amyloid in nature and situated mainly in the anterior and middle stroma. We observed a novel LCD family which carried two pathogenic mutations (R124C and A546D) in the TGFBI gene. The phenotypic features were apparently different from those associated with corresponding single mutations. The result reveals that although the definite mutation is the most important genetic cause of the disease, some different modifier alleles may influence the phenotype.

Role of the GacS Sensor Kinase in the Regulation of Volatile Production by Plant Growth-Promoting Pseudomonas fluorescens SBW25.

In plant-associated Pseudomonas species, the production of several secondary metabolites and exoenzymes is regulated by the GacS/GacA two-component regulatory system (the Gac-system). Here, we investigated if a mutation in the GacS sensor kinase affects the production of volatile organic compounds (VOCs) in P. fluorescens SBW25 (Pf.SBW25) and how this impacts on VOCs-mediated growth promotion and induced systemic resistance of Arabidopsis and tobacco. A total of 205 VOCs were detected by Gas Chromatography Mass Spectrometry for Pf. SBW25 and the gacS-mutant grown on two different media for 3 and 6 days. Discriminant function analysis followed by hierarchical clustering revealed 24 VOCs that were significantly different in their abundance between Pf.SBW25 and the gacS-mutant, which included three acyclic alkenes (3-nonene, 4-undecyne, 1-undecene). These alkenes were significantly reduced by the gacS mutation independently of the growth media and of the incubation time. For Arabidopsis, both Pf.SBW25 and the gacS-mutant enhanced, via VOCs, root and shoot biomass, induced systemic resistance against leaf infections by P. syringae and rhizosphere acidification to the same extent. For tobacco, however, VOCs-mediated effects on shoot and root growth were significantly different between Pf.SBW25 and the gacS-mutant. While Pf.SBW25 inhibited tobacco root growth, the gacS-mutant enhanced root biomass and lateral root formation relative to the non-treated control plants. Collectively these results indicate that the sensor kinase GacS is involved in the regulation of VOCs production in Pf.SBW25, affecting plant growth in a plant species-dependent manner.

Gac-mediated changes in pyrroloquinoline quinone biosynthesis enhance the antimicrobial activity of Pseudomonas fluorescens SBW25.

In Pseudomonas species, production of secondary metabolites and exoenzymes is regulated by the GacS/GacA two-component regulatory system. In Pseudomonas fluorescens SBW25, mutations in the Gac-system cause major transcriptional changes and abolished production of the lipopeptide viscosin and of an exoprotease. In contrast to many other Pseudomonas species and strains, inactivation of the Gac-system in strain SBW25 significantly enhanced its antimicrobial activities against oomycete, fungal and bacterial pathogens. Here, random plasposon mutagenesis of the gacS mutant led to the identification of seven mutants with reduced or loss of antimicrobial activity. In four mutants, the plasposon insertion was located in genes of the pyrroloquinoline quinone (PQQ) biosynthesis pathway. Genetic complementation, ectopic expression, activity bioassays and Reversed-phase high-performance liquid chromatography (RP-HPLC) analyses revealed that a gacS mutation in SBW25 leads to enhanced expression of pqq genes, resulting in an increase in gluconic and 2-ketogluconic acid production, which in turn acidified the extracellular medium to levels that inhibit growth of other microorganisms. We also showed that PQQ-mediated acidification comes with a growth penalty for the gacS mutant in the stationary phase. In conclusion, PQQ-mediated acidification compensates for the loss of several antimicrobial traits in P. fluorescens SBW25 and may help gac mutants to withstand competitors.

Abstract 4267: Quantification of somatic hotspot mutations in KRAS, HRAS, BRAF, and PIK3CA: Comparing normal human breast and ductal carcinoma

Abstract Data within the Catalogue of Somatic Mutations in Cancer indicates that a number of different hotspot oncogene mutations occur infrequently in breast tumors as compared to other tumor types (e.g., colon and lung). We hypothesized that greater sensitivity is required to detect somatic mutations in breast tumors, due to their highly-heterogeneous clonal structure. Therefore, the Allele-specific Competitive Blocker PCR method (with a sensitivity of 10-5), was used to quantify mutations in DNA isolated from normal human breast and ductal carcinoma (DC) samples (n = 10). The specific mutations quantified were: KRAS codon 12 GGT to GAT (G12D) and GGT to GTT (G12V), HRAS codon 12 GGC to GAC (G12D), BRAF codon 600 GTG to GAG (V600E), and PIK3CA codon 1047 CGT to CAT (H1047R). In addition, comparison of normal and malignant tissue was employed in order to define the lower bound on abnormal levels of mutation, as tumors that possess a mutant fraction (MF) greater than the upper 95% confidence interval of that present in normal breast. Interestingly, different patterns of mutation were observed for the different hotspot point mutations. We previously demonstrated that significantly higher levels of KRAS mutation are present in colon and lung tumors relative to the cognate normal tissue; however, the frequencies of KRAS codon 12 GGT to GAT and GGT to GTT mutation in normal breast and in DCs were quite similar (∼10-5 for the KRAS codon 12 GGT to GAT mutation in both tissues). Only one normal sample and two DCs had BRAF codon 600 GTG to GAG MFs >10-5. With respect to HRAS codon 12 GGC to GAC mutation, however, ∼80% of DCs had a MF greater than the upper 95% confidence interval of that present in normal breast tissue. Importantly, no breast tumor had a HRAS codon 12 GGC to GAC MF ≥10-1 (i.e., that detectable by DNA sequencing). Unexpectedly high levels of PIK3CA codon 1047 CGT to CAT mutation (>10-2) were observed in 4/10 normal breast samples and 4/10 DCs, suggesting that preexisting PIK3CA mutations in normal breast contribute to breast cancer susceptibility. In summary, all DC samples analyzed contained somatic mutations, none of which would be detectable by DNA sequencing. Consequently, these results indicate somatic mutations are more prevalent in breast cancer than is currently recognized. Ongoing analyses of additional tumors, corresponding to different breast cancer subtypes, will provide a better understanding of the role of somatic mutations in breast cancer. Citation Format: Malathi Banda, Meagan B. Myers, Karen L. McKim, Yiying Wang, Barbara L. Parsons. Quantification of somatic hotspot mutations in KRAS, HRAS, BRAF, and PIK3CA: Comparing normal human breast and ductal carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4267. doi:10.1158/1538-7445.AM2014-4267

The Sensor Kinase GacS Negatively Regulates Flagellar Formation and Motility in a Biocontrol Bacterium, Pseudomonas chlororaphis O6.

The GacS/GacA two component system regulates various traits related to the biocontrol potential of plant-associated pseudomonads. The role of the sensor kinase, GacS, differs between strains in regulation of motility. In this study, we determined how a gacS mutation changed cell morphology and motility in Pseudomonas chlororaphis O6. The gacS mutant cells were elongated in stationary-phase compared to the wild type and the complemented gacS mutant, but cells did not differ in length in logarithmic phase. The gacS mutant had a two-fold increase in the number of flagella compared with the wild type strain; flagella number was restored to that of the wild type in the complemented gacS mutant. The more highly flagellated gacS mutant cells had greater swimming motilities than that of the wild type strain. Enhanced flagella formation in the gacS mutant correlated with increased expression of three genes, fleQ, fliQ and flhF, involved in flagellar formation. Expression of these genes in the complemented gacS mutant was similar to that of the wild type. These findings show that this root-colonizing pseudomonad adjusts flagella formation and cell morphology in stationary-phase using GacS as a major regulator.

First North American case of Hemoglobin Shepherds Bush (β 74[E18] Gly → Asp) in a central Pennsylvania family

BackgroundHemoglobin Shepherds Bush (Human Genome Variation Society name: HBB:c.224G > A) is an unstable hemoglobin variant resulting from a β 74 GGC to GAC mutation (Gly to Asp) that manifests clinically as hemolytic anemia or gall bladder disease due to chronic subclinical hemolysis.Case presentationWe report a Pennsylvania family of English descent with this condition, first noticed in a 6-year-old female. The proband presented with splenomegaly, fatigue, dark urine and an elevated indirect bilirubin. Hemoglobin identification studies and subsequent genetic testing performed according to a systematic algorithm elucidated the diagnosis of Hb Shepherds Bush.ConclusionsThis is the first case of this rare hemoglobin variant identified in North America to our knowledge. It was identified using a systematic algorithm of diagnostic tests that should be followed whenever considering a rare hemoglobinopathy as part of the differential diagnosis.

Fannin-Lubbock-I [α₂β₂¹¹⁹(GLY>ASP)], a rare mutation in the beta-globin gene, has been detected for the first time in a Hindu Brahmin family in West Bengal, India.

This study aims to describe the hemoglobin Fannin-Lubbock-I, which has a rare mutation substituting the amino acid glycine with aspartic acid at codon 119 of the β-globin chain. A Bengalee Hindu Brahmin family from Kolkata in West Bengal was the focus of this study. Molecular analysis using ARMS-PCR and direct DNA sequencing revealed the presence of a GGC > GAC mutation in codon 119 of the β-globin gene in a heterozygote state in three women of the same family. This is the first report of the hemoglobin Fannin-Lubbock-I from India. Our results will help to identify this mutation, which is relatively infrequent in our population.

HB Fannin-Lubbock-I with A Single GGC>GAC Mutation at β119(GH2)Gly→Asp in a Homozygous Mexican Patient

We studied a fast-moving, abnormal hemoglobin (Hb) identified as Fannin–Lubbock-I [β119(GH2)Gly→Asp] in a homozygous Mexican girl. To date, homozygosity for the Hb Fannin-Lubbock-I variant has not been reported. Her parents and five other relatives were heterozygotes. The 5′ β-globin haplotype analysis showed that the mutation was associated with haplotype 2 [− + + − +]for the ε, Gγ, Aγ, 5′ and 3 ′ψβ-globin sites, and also segregated with the TGTTC haplotype, which was constructed with five polymorphic sites of the β-globin gene [exon 1-nucleotide (nt) 6 (C>T) and IVS-II-16 (C>G), IVS-II-46 (T>C), IVS-II-74 (G>T), and IVS-II-81 (C>T). In 1994, a variant with an additional mutation at codon 111 [β111(G13)Val→Leu] was described in five Spanish families. This variant was termed Hb Fannin-Lubbock-II, and the question of the existence of Hb Fannin-Lubbock-I arose. However, based on our findings, we were able to confirm the existence of Hb Fannin-Lubbock-I and propose that this mutation has a different origin from the one identified in Spanish families.

Temperature-responsive sensing regulates biocontrol factor expression in Pseudomonas fluorescens CHA0

In the plant-beneficial, root-colonizing strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively regulates the synthesis of biocontrol factors (mostly antifungal secondary metabolites) and contributes to oxidative stress response via the stress sigma factor RpoS. The backbone of this pathway consists of the GacS/GacA two-component system, which activates the expression of three small regulatory RNAs (RsmX, RsmY, RsmZ) and thereby counters translational repression exerted by the RsmA and RsmE proteins on target mRNAs encoding biocontrol factors. We found that the expression of typical biocontrol factors, that is, antibiotic compounds and hydrogen cyanide (involving the phlA and hcnA genes), was significantly lower at 35 degrees C than at 30 degrees C. The expression of the rpoS gene was affected in parallel. This temperature control depended on RetS, a sensor kinase acting as an antagonist of the GacS/GacA system. An additional sensor kinase, LadS, which activated the GacS/GacA system, apparently did not contribute to thermosensitivity. Mutations in gacS or gacA were epistatic to (that is, they overruled) mutations in retS or ladS for expression of the small RNAs RsmXYZ. These data are consistent with a model according to which RetS-GacS and LadS-GacS interactions shape the output of the Gac/Rsm pathway and the environmental temperature influences the RetS-GacS interaction in P. fluorescens CHA0.

Spontaneous Mutation of Hemoglobin Lufkin in a White Boy

A 10-year-old white boy presented clinically with thalassemia major facies, pallor, jaundice, and hepatomegaly. Investigation revealed the patient has hemoglobin (Hb) Lufkin concurrent with beta(0) thalassemia. DNA sequencing of the beta globin gene confirmed a GGC to a GAC mutation at codon 29 (gly to asp) for Hb Lufkin on the patient and also revealed a beta(0) thalassemia mutation, IVS-1-1 (G to A), on both the patient and his mother. Both parents lack the Hb Lufkin mutation. Molecular studies, human leukocyte antigen, and red blood cells phenotypic studies indicate spontaneous mutation of Hb Lufkin in this patient.

A GacS deficiency does not affectPseudomonas chlororaphisPA23 fitness when growing on canola, in aged batch culture or as a biofilm

Pseudomonas chlororaphis PA23 is a biocontrol agent that protects against the fungal pathogen Sclerotinia sclerotiorum. Employing transposon mutagenesis, we isolated a gacS mutant that no longer exhibited antifungal activity. Pseudomonas chlororaphis PA23 was previously reported to produce the nonvolatile antibiotics phenazine 1-carboxylic acid and 2-hydroxyphenazine. We report here that PA23 produces additional compounds, including protease, lipase, hydrogen cyanide, and siderophores, that may contribute to its biocontrol ability. In the gacS mutant background, generation of these products was markedly reduced or delayed with the exception of siderophores, which were elevated. Not surprisingly, this mutant was unable to protect canola from disease incited by S. sclerotiorum. The gacS mutant was able to sustain itself in the canola phyllosphere, therefore, the loss of biocontrol activity can be attributed to a reduced production of antifungal compounds and not a declining population size. Competition assays between the mutant and wild type revealed equivalent fitness in aged batch culture; consequently, the gacS mutation did not impart a growth advantage in the stationary phase phenotype. Under minimal nutrient conditions, the gacS-deficient strain produced a tenfold less biofilm than the wild type. However, no difference was observed in the ability of the mutant biofilm to protect cells from lethal antibiotic challenge.

Multiple cutaneous neuromas and macular amyloidosis associated with medullary thyroid carcinoma

Multiple cutaneous neuromas are rarely seen in dermatology practice. We report a case of multiple cutaneous neuromas, macular amyloidosis (MA), and medullary thyroid carcinoma (MTC) and discuss the interrelationship of the associated conditions. Multiple endocrine neoplasia 2 (MEN 2) is a hereditary syndrome that comprises MEN 2A, MEN 2B, and familial MTC. Germline mutations in the RET proto-oncogene is the underlying cause of the syndrome. MEN 2A and MEN 2B show some common endocrine manifestations including MTC and pheochromocytoma. There are reports of families with MA and MEN 2A. Multiple mucosal neuromas occur in 100% of patients with MEN 2B syndrome. Cutaneous neuromas are infrequently reported in MEN 2B syndrome. Our patient was a heterozygote carrier of GAG-->GAC mutation (Glu 768 Asp) in exon 13, codon 768 of the RET proto-oncogene. We speculate that our patient may represent an unusual presentation of MEN 2B or an overlap of MEN 2A and MEN 2B syndromes or a sporadic MTC case with unusual associations.

The GacS sensor kinase controls phenotypic reversion of small colony variants isolated from biofilms of Pseudomonas aeruginosa PA14

The GacS/GacA two-component regulatory system in pseudomonads regulates genes involved in virulence, secondary metabolism and biofilm formation. Despite these regulatory functions, some Pseudomonas species are prone to spontaneous inactivating mutations in gacA and gacS. A gacS(-) strain of Pseudomonas aeruginosa PA14 was constructed to study the physiological role of this sensor histidine kinase. This loss-of-function mutation was associated with hypermotility, reduced production of acylhomoserine lactones, impaired biofilm maturation, and decreased antimicrobial resistance. Biofilms of the gacS(-) mutant gave rise to phenotypically stable small colony variants (SCVs) with increasing frequency when exposed to silver cations, hydrogen peroxide, human serum, or certain antibiotics (tobramicin, amikacin, azetronam, ceftrioxone, oxacilin, piperacillin or rifampicin). When cultured, the SCV produced thicker biofilms with greater cell density and greater antimicrobial resistance than did the wild-type or parental gacS(-) strains. Similar to other colony morphology variants described in the literature, this SCV was less motile than the wild-type strain and autoaggregated in broth culture. Complementation with gacS in trans restored the ability of the SCV to revert to a normal colony morphotype. These findings indicate that mutation of gacS is associated with the occurrence of stress-resistant SCV cells in P. aeruginosa biofilms and suggests that in some instances GacS may be necessary for reversion of these variants to a wild-type state.

Thiamine-auxotrophic mutants of Pseudomonas fluorescens CHA0 are defective in cell-cell signaling and biocontrol factor expression.

In the biocontrol strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to N-acyl-homoserine lactones. Transposon Tn5 was used to isolate P. fluorescens CHA0 insertion mutants that expressed an rsmZ-gfp fusion at reduced levels. Five of these mutants were gacS negative, and in them the gacS mutation could be complemented for exoproduct and signal synthesis by the gacS wild-type allele. Furthermore, two thiamine-auxotrophic (thiC) mutants that exhibited decreased signal synthesis in the presence of 5 x 10(-8) M thiamine were found. Under these conditions, a thiC mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a thiC mutant was impaired for biological control of Pythium ultimum on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by P. fluorescens.

Ten Year-Old Male with Hemoglobin Lufkin/Beta-Zero Thalassemia Compound Heterozygote Who Exhibited Beta-Thalassemia Major Characteristics.

Hemoglobin Lufkin is a rare and mildly unstable hemoglobin variant with increased oxygen affinity. Since 1977, two cases of hemoglobin Lufkin trait and one hemoglobin Lufkin/hemoglobin S have been described. This report is the first case of hemoglobin Lufkin/beta-zero thalassemia in a 10 year-old Caucasian male of Irish/Italian/German background. The patient presented with jaundice, splenomegaly and thalassemia major facies. On blood smear examination, RBC morphology showed hypochromia, microcytosis, many target cells, some spherocytes and basophilic stippling of RBC's. On both alkaline hemoglobin (Hb) electrophoresis and isoelectric focusing (IEF) there was an absence of Hb A with a predominant band slightly anodal to the Hb A position. Hb A2 (4.8%)and Hb F (5.3%) were elevated as measured by high performance liquid chromatography. The heat unstable Hb test was abnormal. DNA sequencing of the beta globin gene confirmed a GCC to GAC mutation at codon 29 (gly to asp) consistent with Hb Lufkin. DNA sequence analysis also revealed a beta-zero thalassemia mutation, IVS-1-1, (G to A). The mother's sample also showed the same beta-thalassemia mutation. Neither hemoglobin Lufkin nor beta-thalassemia were identified in the father; further studies are being done.

Combined subcarcinogenic benzo[a]pyrene and UVA synergistically caused high tumor incidence and mutations in H‐ras gene, but not p53, in SKH‐1 hairless mouse skin

Combined subcarcinogenic doses of benzo[a]pyrene (BaP) and UVA induced H-ras, but not p53, gene mutations 8 weeks before tumor emergence in SKH-1 mice. Neither UVA (40 kJ/m2) nor BaP (8 nmol) induced any tumors after mice were topically treated 3 times/week for 25 weeks. However, combined BaP-UVA treatment synergistically increased tumor incidence and multiplicity. All tumors induced by BaP-UVA were malignant. The epidermis was collected from mice treated for 2, 6 and 10 weeks. DNA from UVB- (0.3 kJ/m2) or BaP-UVA-(8 nmol and 40 kJ/m2-induced tumors was isolated and screened for H-ras and p53 mutations. Four types of point mutation, GGC-->GAC, GCC, GTC and CGC, occurred in UVB-induced tumors at H-ras codon 13; and one type of point mutation, GGA-->GAA, at codon 12. Treatment with either BaP alone or BaP-UVA for 10 weeks caused GGA-->GAA mutation at codon 12 or GGC-->GAC mutation at codon 13 in nontumor skin, respectively, as well as in tumors induced by BaP-UVA. All of the 10-week samples treated with either BaP or BaP-UVA showed detectable mutations at codons 12 and 13, but the genetic load was significantly higher in BaP-UVA-treated mice than in those exposed only to BaP. UVA alone induced mutations at codon 12 in only one-third of samples. G-->A mutations induced by BaP or BaP-UVA at position 38 of codon 13 have not been reported previously. C-->T transitions were detected in p53 hot spots of exon 8 in 2 of 19 BaP-UVA-induced tumors but were not found in nontumor skin.

Molecular Nature of Spontaneous Modifications ingacSWhich Cause Colony Phase Variation inPseudomonassp. 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.

Posttranscriptional repression of GacS/GacA-controlled genes by the RNA-binding protein RsmE acting together with RsmA in the biocontrol strain Pseudomonas fluorescens CHA0.

In the plant-beneficial soil bacterium Pseudomonas fluorescens CHA0, the production of biocontrol factors (antifungal secondary metabolites and exoenzymes) is controlled at a posttranscriptional level by the GacS/GacA signal transduction pathway involving RNA-binding protein RsmA as a key regulatory element. This protein is assumed to bind to the ribosome-binding site of target mRNAs and to block their translation. RsmA-mediated repression is relieved at the end of exponential growth by two GacS/GacA-controlled regulatory RNAs RsmY and RsmZ, which bind and sequester the RsmA protein. A gene (rsmE) encoding a 64-amino-acid RsmA homolog was identified and characterized in strain CHA0. Overexpression of rsmE strongly reduced the expression of target genes (hcnA, for a hydrogen cyanide synthase subunit; aprA, for the main exoprotease; and phlA, for a component of 2,4-diacetylphloroglucinol biosynthesis). Single null mutations in either rsmA or rsmE resulted in a slight increase in the expression of hcnA, aprA, and phlA. By contrast, an rsmA rsmE double mutation led to strongly increased and advanced expression of these target genes and completely suppressed a gacS mutation. Both the RsmE and RsmA levels increased with increasing cell population densities in strain CHA0; however, the amount of RsmA showed less variability during growth. Expression of rsmE was controlled positively by GacA and negatively by RsmA and RsmE. Mobility shift assays demonstrated specific binding of RsmE to RsmY and RsmZ RNAs. The transcription and stability of both regulatory RNAs were strongly reduced in the rsmA rsmE double mutant. In conclusion, RsmA and RsmE together account for maximal repression in the GacS/GacA cascade of strain CHA0.

Atypical hemoglobin H disease in a Thai patient resulting from a combination of α-thalassemia 1 and hemoglobin Constant Spring with hemoglobin J Bangkok heterozygosity

A case of hemoglobin H disease in combination with hemoglobin Constant Spring and a beta-globin chain variant is reported in a 3-yr-old Thai girl. On routine cellulose acetate electrophoresis, one abnormal band in addition to the hemoglobins A, A2, H, Bart's and Constant Spring was detected. The amount of this abnormal band movement towards more anodic to the hemoglobin A was 35.7%. DNA analysis of the alpha-globin gene cluster by polymerase chain reaction (PCR) revealed a combination of defects caused by the SEA-type alpha-thalassemia 1 and the alpha-Constant Spring gene. Analysis of beta-globin gene by PCR and DNA sequencing also detected the heterozygosity for the GGC-GAC mutation at codon 56, leading to a substitution of aspartic acid for glycine resulting in the hemoglobin J Bangkok. The hematologic data of this unusual case of hemoglobin H disease are presented and compared with two compound heterozygotes for hemoglobin J Bangkok and alpha-thalassemia 1 found in the patient's father and grandfather. A simple DNA assay based on an allele-specific PCR for rapid diagnosis of the hemoglobin J Bangkok is also described.