Strain Q1 Research Articles

Compound Sodium Nitrophenolate Promotes Denitrification by Nitrifying Bacteria by Upregulating Nitrate Reductase

Biological denitrification is an efficient and low-cost method to treat wastewater, and it has been shown that growth promoters can regulate the metabolism of microorganisms. This study aimed to investigate the effects of gibberellic acid, naphthalene acetic acid, compound sodium nitrophenolate, and diethyl aminoethyl hexanoate on the growth and denitrification process of denitrifying microorganisms and to examine the associated mechanisms. All four tested growth promoters did not affect the growth of the strain Q1; further, compound sodium nitrophenolate could significantly improve the bacterial denitrification efficiency and showed an increase in the removal rate of 13.08% in 72 h. The addition of 15 mg/L compound sodium nitrophenolate increased the removal rate of strain Q1 by 25.88% at 72 h, significantly improving the efficiency of reducing the chemical oxygen demand of the effluent. Transcriptome analysis identified 1664 differentially expressed genes (573 upregulated and 1091 downregulated genes) in the strain Q1 treated with compound sodium nitrophenolate. Nitrate reductase and nitrate transporter, which are two key enzymes related to the nitrate reduction pathway, were found to be upregulated during the denitrification process. Compound sodium nitrophenolate has promising applications in high-salt and high-nitrogen wastewater treatment.

First report of Pseudomonas palleroniana causing potato soft rot in China.

Slimy potato (Solanum tuberosum) tubers with a foul-smelling odor were obtained from a potato field during harvest in Huize county (25.94°N; 103.40°E), Yunnan province, China in August 2021. The incidence of symptomatic potato tubers was approximately 5% while no symptoms were observed on potato stems. To isolate the causal agent, potato tubers were surface-disinfected and infected tissues were mashed in a 15 ml sterile centrifuge tube with 2 ml sterile distilled water. After a series of dilutions, a volume of 200 μl bacterial suspension from each dilution was spread on nutrient agar (NA) medium and incubated at 28 °C for 48 h. Single colonies randomly selected from NA plates were then picked and separately subcultured in nutrient broth (NB) medium. Pure cultures were acquired by successive streaking on NA medium. The colonies with irregular shapes were white and opaque (Fig. S1A). Among all the isolates, strain Q1 exhibited pectinolytic activity on inoculated potato tubers using a pin prick method with an inoculum concentration of 108 CFU/ml while no symptoms appeared for the control group using sterile water (Fig. S1B). However, no obvious pits were observed for strain Q1 when tested on crystal violet pectin (CVP) medium. To further identify the strain Q1, total DNA was extracted using the TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit. PCR amplification of 16S rDNA of strain Q1 was performed using the universal PCR primer pair 27F/1492R and followed by Sanger sequencing. The BLASTn analysis of the sequence (NCBI accession number ON631256) based on NCBI rRNA/ITS databases revealed 100% query coverage and 99.78% identity to the 16S rDNA sequences of type strain Pseudomonas tolaasii ATCC 33618 and Pseudomonas palleroniana CFBP 4389, respectively. Whole-genome sequencing of strain Q1 was then performed using the Illumina and Nanopore sequencing platform. A single contig (NCBI accession number CP092411) with a length of approx. 6.2 MB was obtained by de novo assembly using Raven (v1.5.1) (Vaser and Šikić 2021) and Pilon (v1.24) (Walker et al. 2014). The completeness and redundancy of this assembly was evaluated by BUSCO (v5.2.2) (Manni et al. 2021), and a score of 100% completeness indicated a high quality of the assembly. The genome sequence of strain Q1 was uploaded to the Type Strain Genome Server (TYGS) for a whole genome-based taxonomic analysis (Meier-Kolthoff and Göker 2019). The distance-based phylogeny revealed that strain Q1 is well clustered together with Pseudomonas palleroniana (Fig. S1C). Furthermore, a digital DNA-DNA hybridization (dDDH) value (86.9%) between strain Q1 and P. palleroniana was above the species boundary (≥70%), indicating that strain Q1 should be classified as P. palleroniana. In addition, P. palleroniana was re-isolated from potato tubers inoculated using a pin prick method with an inoculum concentration of 108 CFU/ml and was identified by morphological similarities and 16s rDNA sequencing described above, thus fulfilling Koch's postulates. Furthermore, aerial stem rot symptoms were observed after 3-5 days post inoculation of strain Q1 with a concentration of 108 CFU/ml on stems of young potato plants using a pin prick method (Fig. S1D). Notably, a couple of Pseudomonas spp. have been reported to cause soft rot in carrot (Godfrey and Marshall 2002) and melon (Zhang et al. 2016). To our knowledge, this is the first report of P. palleroniana causing soft rot on potato tuber in China.

Host immune response to infectious bronchitis virus Q1 in two commercial broiler chicken lines

This study investigated the pathogenesis of infectious bronchitis virus (Gammacoronavirus) strain Q1 in two commercial broiler chicken lines, and the host immune response to infection. Chicks from each line were grouped into either infected or control. Following Q1 infection at day-old, fast (Line-A) and slow (Line-B) growing chicks were monitored for clinical signs and body weights. At 3, 7, 9, 14, 21 and 28 days post infection (dpi), five birds were humanely euthanised, and trachea, kidney and proventriculus tissues were collected for quantitative RT-PCR and histopathology. Blood was collected weekly to determine IBV-specific ELISA antibody titres. Q1 infection significantly reduced the body weights of Line-A chicks at 14 and 21 dpi, but there were no significant differences in Line-B. Through qRT-PCR, significantly higher viral loads were found in the trachea, proventriculus and kidney tissues of Line-A chicks at 7–9 dpi. At day-old and at 28 dpi, the mean antibody titre in Line-B was notably higher than Line-A. Significant IFN-α mRNA expression was noted in the trachea and kidneys of Line-A, whereas no change occurred in Line-B. Chicks in Line-B, compared to those in Line-A, demonstrated a tissue-dependent increase of IFN-β, TLR3, IL-1β and IL-6 and LITAF gene transcription responses to IBV Q1. It appears that the level of maternal antibodies, growth rates, and other inherent host genetic factors could have influenced the differences in viral loads and immune responses.

Biochemical characterisation of a collagenase from Bacillus cereus strain Q1

Collagen is the most abundant protein in higher animals and as such it is a valuable source of amino acids and carbon for saprophytic bacteria. Due to its unique amino acid composition and triple-helical tertiary structure it can however only be cleaved by specialized proteases like the collagenases secreted by some bacteria. Among the best described bacterial collagenases are ColG and ColH from Clostridium histolyticum. Many Bacillus species contain homologues of clostridial collagenases, which play a role in some infections caused by B. cereus. Detailed biochemical and enzymatic characterizations of bacillial collagenases are however lacking at this time. In an effort to close this gap in knowledge we expressed ColQ1 from B. cereus strain Q1 recombinantly, investigated its metal dependency and performed peptide, gelatin and collagen degradation assays. Our results show that ColQ1 is a true collagenase, cleaving natively folded collagen six times more efficiently than ColG while at the same time being a similarly effective peptidase as ColH. In both ColQ1 and ColG the rate-limiting step in collagenolysis is the unwinding of the triple-helix. The data suggest an orchestrated multi-domain mechanism for efficient helicase activity.

Exogenous ACC Deaminase Is Key to Improving the Performance of Pasture Legume-Rhizobial Symbioses in the Presence of a High Manganese Concentration.

Manganese (Mn) toxicity is a very common soil stress around the world, which is responsible for low soil fertility. This manuscript evaluates the effect of the endophytic bacterium Pseudomonas sp. Q1 on different rhizobial-legume symbioses in the absence and presence of Mn toxicity. Three legume species, Cicer arietinum (chickpea), Trifolium subterraneum (subterranean clover), and Medicago polymorpha (burr medic) were used. To evaluate the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase produced by strain Q1 in these interactions, an ACC deaminase knockout mutant of this strain was constructed and used in those trials. The Q1 strain only promoted the symbiotic performance of Rhizobium leguminosarum bv. trifolii ATCC 14480T and Ensifer meliloti ATCC 9930T, leading to an increase of the growth of their hosts in both conditions. Notably, the acdS gene disruption of strain Q1 abolished the beneficial effect of this bacterium as well as causing this mutant strain to act deleteriously in those specific symbioses. This study suggests that the addition of non-rhizobia with functional ACC deaminase may be a strategy to improve the pasture legume–rhizobial symbioses, particularly when the use of rhizobial strains alone does not yield the expected results due to their difficulty in competing with native strains or in adapting to inhibitory soil conditions.

In situ Listeria monocytogenes biocontrol and sensory attributes enhancement in raw beef meat by Enterococcus lactis

The enterocin P-producing Enterococcus lactis strain Q1 was directly added at 107 CFU/g in refrigerated raw beef to evaluate its effectiveness in the control of Listeria monocytogenes EGDe 107776 proliferation. Furthermore, sensorial evaluation of the quality of treated beef meat samples with the enterococcal protective culture was performed basing on color, odor, and appearance parameters for 14 days. As a result, it was demonstrated that the enterocin P-producing E. lactis Q1 strain led to a remarkable reduction (p < .05) of L. monocytogenes in beef meat by 6.47 ± 0.30 log units after 7 days and 7.25 ± 0.35 log units below the control after 14 days, then remained stable up to 28 days of cold storage. Also, E. lactis Q1 strain improved significantly (p < .05) the organoleptic quality of treated beef sample. Hence, E. lactis Q1 clearly indicates its efficiency to be used as protective agent in a wide range of meat products. Practical applications Preserving meat using new bacteriocinogenic lactic acid bacteria is an important agenda to ensure consumer safety. The present study showed the ability of an enterocin P-producing strain named E. lactis Q1, added in raw beef meat during cold storage, to inhibit L. monocytogenes and improve meat sensory properties. Hence, E. lactis Q1 can be used in the future to develop healthy meat products with better shelf life and quality.

Lipolytic bacterial strains mediated transesterification of non-edible plant oils for generation of high quality biodiesel

Biodiesel is one of the best alternative to depleting fossil fuels for transport sector. However, biodiesel production via lipase mediated transesterification has limitation of high costing microbial enzymes. In order to overcome this limitation, a process of sequential treatment of oil industry wastewater using isolated lipolytic bacterial strains and biodiesel production from non-edible plant oils was studied. In this study, efficient lipase producing bacteria were isolated and evaluated for production of biodiesel from mustard, soybean, jatropha and taramira oils utilizing methanol for the transesterification of oils and bioremediation. Selected strains were then identified, using 16s rRNA sequencing. Further, Bacillus subtilis strain Q1 KX712301 was optimized for biodiesel production from non-edible taramira oil via Plackett-Burman and central composite design. Highest volumetric yield of biodiesel obtained was 102% at optimized parameters. Finally, a sequential bioremediation of vegetable oil contaminated wastewater and then microbial production of biodiesel from non-edible taramira oil was carried out using efficient lipase producer B.subtilis strain Q1 at optimized conditions. During sequential process, complete chemical oxigen demand reduction of oil containing wastewater and theoretical volumetric yield of biodiesel was achieved. Gas chromatography/mass spectrometry chromatogram revealed that the total fatty acid methyl ester content of the produced biodiesel was >98% which is in accordance with the biodiesel quality standards specified by both ASTM and EU-14103.

Discovery of a Potent Inhibitor Class with High Selectivity toward Clostridial Collagenases.

Secreted virulence factors like bacterial collagenases are conceptually attractive targets for fighting microbial infections. However, previous attempts to develop potent compounds against these metalloproteases failed to achieve selectivity against human matrix metalloproteinases (MMPs). Using a surface plasmon resonance-based screening complemented with enzyme inhibition assays, we discovered an N-aryl mercaptoacetamide-based inhibitor scaffold that showed sub-micromolar affinities toward collagenase H (ColH) from the human pathogen Clostridium histolyticum. Moreover, these inhibitors also efficiently blocked the homologous bacterial collagenases, ColG from C. histolyticum, ColT from C. tetani, and ColQ1 from the Bacillus cereus strain Q1, while showing negligible activity toward human MMPs-1, -2, -3, -7, -8, and -14. The most active compound displayed a more than 1000-fold selectivity over human MMPs. This selectivity can be rationalized by the crystal structure of ColH with this compound, revealing a distinct non-primed binding mode to the active site. The non-primed binding mode presented here paves the way for the development of selective broad-spectrum bacterial collagenase inhibitors with potential therapeutic application in humans.

Isolation and characterisation of an enterocin P-producing Enterococcus lactis strain from a fresh shrimp (Penaeus vannamei).

Screening for lactic acid bacteria (LAB) from fresh shrimp samples (Penaeus vannamei) collected from retail seafood markets in the Tunisian's coast, resulted in the isolation of an Enterococcus strain termed Q1. This strain was selected for its antagonistic activity against pathogenic bacteria such as Listeria monocytogenes, Pseudomonas aeruginosa, Lactococcus garvieae and against fungi (Aspergillus niger and Fusarium equiseti). The Q1 strain was characterised using standard morphological and biochemical tests, growth assays at different temperatures, pH and salinity. 16S rRNA, rpoA and pheS gene sequencing, as well as the 16S-23S rRNA intergenic spacer analyses, were combined to identify strain Q1 as a strain of Enterococcus lactis. The bacteriocin produced by E. lactis Q1 is thermostable, active in the pH range from 4.0 to 9.0 and has a bactericidal mode of action. The enterocin P structural gene was detected by specific PCR in strain E. lactis Q1, which is in good agreement with SDS-PAGE data of the purified bacteriocin. A lack of significant antibiotic resistance genes and virulence determinants was confirmed by specific PCRs. This work provides the first description of an enterocin P producer E. lactis strain isolated from a fresh shrimp. Based on its safety properties (absence of haemolytic activity, virulence factors and antibiotic resistance genes), this strain has the potential to be used as a natural additive or adjunct protective culture in food biopreservation and/or probiotic culture.

Effects of phosphate solubilization and phytohormone production of Trichoderma asperellum Q1 on promoting cucumber growth under salt stress

Salinity is one of the major abiotic stresses limiting crop growth and yield. This study investigated the underlying mechanisms of Trichoderma asperellum Q1 in promoting cucumber growth under salt stress, including the abilities of the strain to solubilize phosphate and to produce phytohormone. The results showed that T. asperellum Q1 could solubilize inorganic or organic phosphate and the activities of phosphatases and phytase could be detected in the culture supernatant. In hydroponic experiments, the growth of cucumber seedlings was increased in the hydroponic system treated by culture filtrate of strain Q1 with tricalcium phosphate or calcium phytate under salt stress. This strain also exhibited the ability to produce indole acetic acid (IAA), gibberellic acid (GA) and abscisic acid (ABA) in liquid medium without any inducers. The levels of those three phytohormones in cucumber seedling leaves also increased after inoculated with this strain, along with increased root growth and root activities of the plant. These results demonstrated the mechanisms of T. asperellum Q1 in alleviating the suppression effect of salt stress involving the change of phytohormone levels in cucumber plant and its ability of phosphate solubilization.

Catenovulum maritimus sp. nov., a novel agarolytic gammaproteobacterium isolated from the marine alga Porphyra yezoensis Ueda (AST58-103), and emended description of the genus Catenovulum.

A novel agarolytic, Gram-stain negative, heterotrophic, facultatively anaerobic and pale-white pigmented bacterial strain, designated Q1(T), was isolated from the marine alga Porphyra yezoensis Ueda (AST58-103) collected from the coastal area of Weihai, China. The cells are motile by means of peritrichous flagella. The isolate requires NaCl for growth, while seawater is not necessary, and growth occurs optimally at about 30-33 °C, in 1-3 % (w/v) NaCl and at pH 7-7.5. Strain Q1(T) shows oxidase-positive and catalase-negative activities, and possesses the ability to hydrolyse starch and alginate, but not cellulose, gelatin, urea or Tween-80. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain Q1(T) is affiliated with the family Alteromonadaceae within the class Gammaproteobacteria. The isolate, strain Q1(T), is most closely related to Catenovulum agarivorans YM01(T) (94.85 %), with less than 91.2 % sequence similarity to other close relatives with validly published names. The draft genome sequence of strain Q1(T) consists of 62 contigs (>200 bp) of 4,548,270 bp. The genomes of Q1(T) and YM01(T) have an ANI value of 70.7 %, and the POCP value between the two genomes is 64.4 %. The genomic DNA G+C content of strain Q1(T) is 37.9 mol% as calculated from the draft genome sequence. The main isoprenoid quinone is ubiquinone-8. The predominant cellular fatty acids are summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH), C16:0 and C18:1 ω7c. The major polar lipids are phosphatidylethanolamine and phosphatidylglycerol. Based on data from a polyphasic chemotaxonomic, physiological and biochemical study, strain Q1(T) should be classified as a novel species of the genus Catenovulum, for which the name Catenovulum maritimus sp. nov. is proposed. The type strain is Q1(T) (=CICC 10836(T)=DSM 28813(T)).

Isolation and Degradation Kinetics of the Quinoline Degradation Bacterium Strains from Coking Wastewater

An aerobic bacterial strain Q1 capable of utilizing quinoline as sole source of carbon, nitrogen and energy was isolated from activated sludge of a coke plant wastewater treatment process. The morphological properties and the phylogenetic analysis based on 16S rDNA sequences showed strain Q1 belonged to thePseudomonassp.. Another strain Q2 was unable to utilize quinoline as sole source of energy alone can coexist with Q1 when quinoline was sole energy. The coexistence of Q1 and Q2 has synergistic effect to quinoline degradation. The biodegradation tests suggested that the kinetics of quinoline biodegradation by Q1 or the combaination of Q1and Q2 strain can be described by Haldane equation kinetically.

Diagnostic and clinical observation on the infectious bronchitis virus strain Q1 in Italy.

This paper describes the diagnostic and clinical observations of an infectious bronchitis virus (IBV) variant, referred to as Q1, in clinically ill chickens in Italy. This IBV variant was described for the first time in 1998 in China. In the autumn of 2011 it caused a small-scale epidemic in nonvaccinated meat chickens in farms located in Northern Italy. The disease was characterized by increased mortality, kidney lesions and proventriculitis. Histopathological observations confirmed the nephritis and described an unusual erosive/necrotic proventriculitis with infiltration of lymphocytes, plasma cells and heterophils, as well as fibroplasia in the lamina propria. Despite these findings and the isolation of the Q1 IB virus directly from proventricular tissue, further studies are necessary to confirm the role of this IBV strain in the development of proventricular lesions. Phylogenetic analysis revealed that all the IBV isolates were very similar and probably had a common origin. The IBV Q1 variant appears to be now endemic in the North of Italy and at times it is detected in vaccinated backyard and commercial broiler farms. The importance of continuous monitoring in controlling the spread of known or emerging IBV variants is underlined.

Effects of Taishan Pinus massoniana pollen polysaccharide on the subunit vaccine of Proteus mirabilis in birds

Three adjuvants, namely, Taishan Pinus massoniana pollen polysaccharide (TPPPS), white mineral oil (WO) and propolis (PP), were added to the outer membrane protein (OMP) of Proteus mirabilis (P. mirabilis) and their effects were compared. Three hundred 1-day-old chicks were randomly divided into five groups (I–V), with 60 chicks per group, and injected subcutaneously with WO-OMP vaccine (I), PP-OMP vaccine (II), TPPPS-OMP vaccine (III), OMP-only vaccine (IV) and physiological saline (V) at 3, 7 and 12 days old. On days 3, 7, 14, 21, 28, 35, 42 and 49 after the first vaccination, the antibody titers, interleukin-2 levels (IL-2) and T-lymphocyte proliferation rates in the peripheral blood as well as the secreting-type IgA levels (SIgA) in the duodenum were measured. On day 7 after the third vaccination, the chicks were challenged with P. mirabilis strain Q1 and the protective effects of each group were observed. The highest protective rate was observed in group III. Moreover, the antibody titers as well as IL-2, SIgA and T-lymphocyte proliferation rates in this group significantly increased and were significantly higher than those in the other groups at most time points. The results indicate that TPPPS could significantly enhance the effects of the subunit vaccine of P. mirabilis; induced stronger humoral, cellular and mucosal immunity as compared with WO and PP; and should be developed as a vaccine adjuvant.

Study of the siderophore‐producing Trichoderma asperellum Q1 on cucumber growth promotion under salt stress

Trichoderma spp. are versatile beneficial fungi which can stimulate growth and plant resistance to biotic and abiotic stresses. In this study, the potential of Trichoderma isolate in promoting the cucumber growth under salt stress and its possible mechanisms were investigated. Strain Q1 was isolated from the rhizosphere of cucumber in greenhouse in China and identified as Trichoderma asperellum based on its morphological features and the molecular phylogenetic analyses. It exhibited some plant growth-promoting attributes of phosphate solubilization, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, auxin and siderophore production. In pot trials, applying strain Q1 to cucumber plant had significantly promoted seedlings growth and alleviated the growth suppression induced by salt stress as confirmed by the changes in growth phenotype and several biochemical and physiological parameters. In solution culture experiments, the growth of cucumber seedlings was increased and the percentage of wilted cucumber seedlings was decreased in the treatment of siderophore-containing culture filtrate (SCF) of strain Q1 with insoluble Fe(3+) under salt stress. These results indicated that T. asperellum Q1 has a real potential to enhance cucumber growth by inducing physiological protection under saline stress, and its siderophores showed sign of alleviating negative effect of salinity and available iron deficiency.

Characterization of Strain Pseudomonas sp. Q1 in Microbial Fuel Cell for Treatment of Quinoline-Contaminated Water

To find new strain in the microbial fuel cell (MFC) for quinoline removal from wastewater and soil, a facultative anaerobic bacterium strain was isolated from the anode of MFC, utilizing quinoline as the carbon source and electron donor. Based on the 16S rRNA sequence analysis, the bacterium strain was Gram-negative and identified as Pseudomonas sp. Q1 according to its morphology and physiochemical properties. The strain was inoculated into a double-chambered MFC using various quinoline concentrations (0, 50, 75, 86, 100, 150, 200 and 300 mg L−1) combining with 300 mg L−1 glucose as the fuel. Results showed that electricity was generated from the MFC, in which quinoline was degraded simultaneously. The values of Coulombic efficiency (CE) increased with the increase of quinoline concentrations from 0 to 100 mg L−1 then decreased with the increase of quinoline concentration from 100 to 300 mg L−1, and the maximum CE 36.7% was obtained at the quinoline concentration of 100 mg L−1. The cyclic voltammetry analysis suggested that the mechanism of electron transfer was through excreting mediators produced by the strain Q1. The MFC should be a potential method for the treatment of quinoline-contaminated water and soil.

Molecular and physiological aspects of alcohol dehydrogenases in the ethanol metabolism of Saccharomyces cerevisiae

The physiological role and possible functional substitution of each of the five alcohol dehydrogenase (Adh) isozymes in Saccharomyces cerevisiae were investigated in five quadruple deletion mutants designated strains Q1-Q5, with the number indicating the sole intact ADH gene. Their growth in aerobic batch cultures was characterised in terms of kinetic and stoichiometric parameters. Cultivation with glucose or ethanol as carbon substrate revealed that Adh1 was the only alcohol dehydrogenase capable of efficiently catalysing the reduction of acetaldehyde to ethanol. The oxidation of produced or added ethanol could also be attributed to Adh1. Growth of strains lacking the ADH1 gene resulted in the production of glycerol as a major fermentation product, concomitant with the production of a significant amount of acetaldehyde. Strains Q2 and Q3, expressing only ADH2 or ADH3, respectively, produced ethanol from glucose, albeit less than strain Q1, and were also able to oxidise added ethanol. Strains Q4 and Q5 grew poorly on glucose and produced ethanol, but were neither able to utilise the produced ethanol nor grow on added ethanol. Transcription profiles of the ADH4 and ADH5 genes suggested that participation of these gene products in ethanol production from glucose was unlikely.

Improved synthesis of Salmonella typhimurium enterotoxin using gene fusion expression systems.

Salmonella enterotoxin (Stn) is a virulence factor in S. typhimurium strain Q1 that causes both fluid secretion in ligated intestinal loops of rabbits and elongation of Chinese hamster ovary (CHO) cells. High-level expression systems are needed to provide Stn in soluble form for detailed study of the biological activity of Stn. To maximize the synthesis and solubility of Stn, we systematically compared the production of native Stn synthesized with a T7 RNA polymerase/promoter system to that of two fusion proteins: glutathione S-transferase::Stn (Gst::Stn) and thioredoxin A::Stn (TrxA::Stn). The latter fusion protein expression systems resulted in a 64-fold increase in Gst::Stn and TrxA::Stn antigen concentration, as measured by specific anti-peptide antibodies in an enzyme-linked immunosorbent assay (ELISA). Most of the toxin derived using these vector systems was insoluble; however, the solubility of the TrxA::Stn antigen increased by at least 50-fold, with a concomitant increase in CHO cell elongation activity. In addition, stn gene expression was enhanced more than 50-fold by addition of 0.2-0.4 M NaCl to Luria-Bertani medium. The biological activity of Stn also was increased in the high-osmolarity medium. Consequently, the expression of stn may be regulated by DNA supercoiling.

Molecular characterization of an enterotoxin from Salmonella typhimurium

In this study, we describe the molecular and antigenic characteristics of a cloned enterotoxin from Salmonella typhimurium strain Q1. The full length Salmonella enterotoxin gene (stn), localized on a 2.8 kb Cla I/PstI DNA fragment, was cloned from a genomic library of Salmonella . Based on nucleotide sequence analysis, the stn gene contained 749 bp that would encode a protein having a molecular size of 29 073. The most unusual feature of the stn gene was the presence of a rare initiation codon (TTG) in lieu of the typical ATG codon, which required site-directed mutagenesis to confirm the precise initiation site. The expression of the stn gene in a bacteriophage T7 RNA polymerase/promoter system was enhanced by introducing a typical ATG start codon and an optimal Shine-Dalgarno sequence upstream of the stn gene by site-directed mutagenesis. The stn gene was located opposite the hydHG operon that regulates labile hydrogenase activity in Salmonella species and Escherichia coli . The overall amino acid sequence of the enterotoxin was quite dissimilar to any other published sequence, including cholera toxin or other adenylate cyclase-activating proteins. However, an intriguing similarity in a small region of the amino acid sequence of Stn was observed with portions of the amino acid sequences from several other protein toxins known to ADP-ribosylate host cell proteins. This region of homology may indicate a conserved motif, within the active site, that is involved in the stimulation of adenylate cyclase activity.

Cloning and expression of the Salmonella enterotoxin gene.

This report examines the genetic basis for Salmonella typhimurium Q1 enterotoxin production. A 918-base-pair XbaI-HincII fragment of plasmid pJM17, composed of cholera toxin (CT) coding sequences (ctxAB), was used as a gene probe. With this probe, the S. typhimurium enterotoxin was identified on a 6.3-kilobase EcoRI-PstI fragment of chromosomal DNA from plasmidless strain Q1. We cloned this 6.3-kilobase fragment into Escherichia coli RR1. The genetic map of the cloned Salmonella enterotoxin (stx) gene was similar but not identical to the CT and E. coli heat-labile enterotoxin genes. By using synthetic oligonucleotides derived from the sequences of CT subunits A (ctxA) and B (ctxB), it was revealed that there were some conserved regions of DNA encoding the enterotoxins of strain Q1 and Vibrio cholerae. Expression of the cloned stx gene in minicells and subsequent Western blot (immunoblot) analysis with CT antitoxin demonstrated that the Salmonella enterotoxin had two or more subunits with molecular sizes of 45, 26, and 12 kilodaltons. Crude cell lysates of E. coli RR1(pCHP4), containing the cloned Salmonella enterotoxin gene, elicited fluid secretion in ligated rabbit intestinal loops and firm induration in rabbit skin. Both of these enterotoxic responses were neutralized by antisera specific for CT. Mucosal tissue from positive intestinal loops contained elevated levels of cyclic AMP. These data suggest some evolutionary relatedness between the enterotoxin genes of S. typhimurium and V. cholerae.