The groEL2 gene, but not groEL1, is required for biosynthesis of the secondary metabolite myxovirescin in Myxococcus xanthus DK1622

Microbiology ◽  
2014 ◽  
Vol 160 (3) ◽  
pp. 488-495 ◽  
Author(s):  
Yan Wang ◽  
Xi Li ◽  
Wenyan Zhang ◽  
Xiuwen Zhou ◽  
Yue-zhong Li
Keyword(s):  
Secondary Metabolite ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Myxococcus Xanthus ◽  
Domain Swapping ◽  
Wild Type ◽  
Zone Of Inhibition ◽  
Knockout Mutant ◽  
E Coli

Myxococcus xanthus DK1622 possesses two copies of the groEL gene: groEL1, which participates in development, and groEL2, which is involved in the predatory ability of cells. In this study, we determined that the groEL2 gene is required for the biosynthesis of the secondary metabolite myxovirescin (TA), which plays essential roles in predation. The groEL2-knockout mutant strain was defective in producing a zone of inhibition and displayed decreased killing ability against Escherichia coli, while the groEL1-knockout mutant strain exhibited little difference from the wild-type strain DK1622. HPLC revealed that deletion of the groEL2 gene blocked the production of TA, which was present in the groEL1-knockout mutant. The addition of exogenous TA rescued the inhibition and killing abilities of the groEL2-knockout mutant against E. coli. Analysis of GroEL domain-swapping mutants indicated that the C-terminal equatorial domain of GroEL2 was essential for TA production, while the N-terminal equatorial or apical domains of GroEL2 were not sufficient to rescue TA production of the groEL2 knockout.

scholarly journals A Carotenoid- and Poly-β-Hydroxybutyrate-Free Mutant Strain of Sphingomonas elodea ATCC 31461 for the Commercial Production of Gellan

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Ang Li ◽  
Tingting Hu ◽  
Hangqi Luo ◽  
Nafee-Ul Alam ◽  
Jiaqi Xin ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Gene Knockout ◽  
Commercial Production ◽  
Gellan Gum ◽  
Wild Type ◽  
Knockout Mutant ◽  
Recovery Method ◽  
Sphingomonas Elodea

ABSTRACT Gellan gum is a microbial exopolysaccharide, produced after aerobic fermentation using the Gram-negative bacterium strain Sphingomonas elodea ATCC 31461. Due to its unique structure and excellent physical characteristics, gellan gum has a broad range of applications in food, pharmaceutical, and other industries where it is used for stabilizing, emulsifying, thickening, and suspending. During the fermentative production of gellan, strain ATCC 31461 also accumulates large amounts of the metabolic by-products yellow carotenoid pigments and poly-β-hydroxybutyrate (PHB), which is decreasing the gellan production and increasing processing costs. A pigment PHB-free mutant was obtained by knocking out the phytoene desaturase gene (crtI) in the carotenoid biosynthetic pathway and the phaC gene, encoding a PHB synthase for the polymerization of PHB. Unfortunately, the double gene knockout mutant produced only 0.56 g liter−1 gellan. Furthermore, blocking PHB and carotenoid synthesis resulted in the accumulation of pyruvate, which reduced gellan production. To elevate gellan production, combined UV irradiation and ethyl methanesulfonate (EMS) mutagenesis treatment were used. A mutant strain with the same level of pyruvate as that of the wild-type strain and higher gellan production was isolated (1.35 g liter−1, 132.8% higher than the double gene knockout mutant and 14.4% higher than the wild-type strain ATCC 31461). In addition, a new gellan gum recovery method based on the new mutant strain was investigated, in which only 30% isopropanol was required, which is twice for the wild-type strains, and the performance of the final product was improved. Thus, the mutant strain could be an ideal strain for the commercial production of gellan. IMPORTANCE A carotenoid- and PHB-free double gene knockout strain mutant was constructed to simplify the purification steps normally involved in gellan production. However, the production of gellan gum was unexpectedly reduced. A mutant with 14.4% higher gellan production than that of the wild-type strain was obtained and isolated after employing UV and EMS combined mutagenesis. Based on this high-yield and low-impurity-producing mutant, a new recovery method requiring less organic solvent and fewer operating steps was developed. This method will effectively reduce the production costs and improve the economic benefits of large-scale gellan production.

scholarly journals Pathogenicity of FtsK mutant of avian pathogenic Escherichia coli

2016 ◽  
Vol 60 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Xiaojing Xu ◽  
Xiang Chen ◽  
Song Gao ◽  
Lixiang Zhao
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Economic Losses ◽  
Wild Type ◽  
Avian Pathogenic Escherichia Coli ◽  
Knockout Mutant ◽  
Pathogenic Escherichia Coli ◽  
Specific Pathogen

Abstract Introduction: Avian pathogenic Escherichia coli (APEC) is a leading cause of extraintestinal infection and heavy economic losses. Imparting immunity after vaccination with live attenuated strain vaccination is an ideal strategy for infection control. This study considers an FtsK knockout mutant strain as a candidate. Material and Methods: An FtsK knockout mutant of APEC strain E058 was constructed and the pathogenicity of the mutant and wild-type strains was further evaluated in chickens. Results: The 50% lethal doses of each strain for one-day-old specific-pathogen-free (SPF) chickens challenged experimentally via trachea were 105.5 and 107.0 colony-forming units (CFU) respectively. Chickens challenged with the wild-type strain exhibited typical signs and lesions of avian colibacillosis, while those inoculated with the mutant strain showed mild pericarditis and pulmonary congestion. The growth rate of the FtsK mutant strain was much slower than the wild-type strain in the heart, spleen, liver, and lung of infected chickens. Conclusion: These results indicated that the APEC FtsK mutant can be attenuated for chickens, and that this mutant has the potential for the development of an APEC vaccine.

scholarly journals Mutations in Genes Involved in the Flagellar Export Apparatus of the Solvent-Tolerant Pseudomonas putida DOT-T1E Strain Impair Motility and Lead to Hypersensitivity to Toluene Shocks

2001 ◽  
Vol 183 (14) ◽  
pp. 4127-4133 ◽  
Author(s):  
Ana Segura ◽  
Estrella Duque ◽  
Ana Hurtado ◽  
Juan L. Ramos
Keyword(s):  
Pseudomonas Putida ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Random Mutagenesis ◽  
Luria Bertani ◽  
Wild Type ◽  
Knockout Mutant ◽  
Flagellar Proteins ◽  
Solvent Tolerant

ABSTRACT Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow in the presence of 1% (vol/vol) toluene in the culture medium. Random mutagenesis with mini-Tn5-′phoA-Km allowed us to isolate a mutant strain (DOT-T1E-42) that formed blue colonies on Luria-Bertani medium supplemented with 5-bromo-4-chloro-3-indolylphosphate and that, in contrast to the wild-type strain, was unable to tolerate toluene shocks (0.3%, vol/vol). The mutant strain exhibited patterns of tolerance or sensitivity to a number of antibiotics, detergents, and chelating agents similar to those of the wild-type strain. The mutation in this strain therefore seemed to specifically affect toluene tolerance. Cloning and sequencing of the mutation revealed that the mini-Tn5-′phoA-Km was inserted within the fliPgene, which is part of the fliLMNOPQRflhBA cluster, a set of genes that encode flagellar structure components. FliP is involved in the export of flagellar proteins, and in fact, theP. putida fliP mutant was nonmotile. The finding that, after replacing the mutant allele with the wild-type one, the strain recovered the wild-type pattern of toluene tolerance and motility unequivocally assigned FliP a function in solvent resistance. An flhB knockout mutant, another gene component of the flagellar export apparatus, was also nonmotile and hypersensitive to toluene. In contrast, a nonpolar mutation at the fliLgene, which encodes a cytoplasmic membrane protein associated with the flagellar basal body, yielded a nonmotile yet toluene-resistant strain. The results are discussed regarding a possible role of the flagellar export apparatus in the transport of one or more proteins necessary for toluene tolerance in P. putida DOT-T1E to the periplasm.

scholarly journals The role of Zur-regulated lipoprotein A in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles in Acinetobacter baumannii

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Nayeong Kim ◽  
Hyo Jeong Kim ◽  
Man Hwan Oh ◽  
Se Yeon Kim ◽  
Mi Hyun Kim ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Mutant Strain ◽  
Antimicrobial Susceptibility ◽  
Type Strain ◽  
Wild Type Strain ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Wild Type ◽  
Bacterial Morphology

Abstract Background Zinc uptake-regulator (Zur)-regulated lipoprotein A (ZrlA) plays a role in bacterial fitness and overcoming antimicrobial exposure in Acinetobacter baumannii. This study further characterized the zrlA gene and its encoded protein and investigated the roles of the zrlA gene in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles (OMVs) in A. baumannii ATCC 17978. Results In silico and polymerase chain reaction analyses showed that the zrlA gene was conserved among A. baumannii strains with 97–100% sequence homology. Recombinant ZrlA protein exhibited a specific enzymatic activity of D-alanine-D-alanine carboxypeptidase. Wild-type A. baumannii exhibited more morphological heterogeneity than a ΔzrlA mutant strain during stationary phase. The ΔzrlA mutant strain was more susceptible to gentamicin than the wild-type strain. Sizes and protein profiles of OMVs were similar between the wild-type and ΔzrlA mutant strains, but the ΔzrlA mutant strain produced 9.7 times more OMV particles than the wild-type strain. OMVs from the ΔzrlA mutant were more cytotoxic in cultured epithelial cells than OMVs from the wild-type strain. Conclusions The present study demonstrated that A. baumannii ZrlA contributes to bacterial morphogenesis and antimicrobial resistance, but its deletion increases OMV production and OMV-mediated host cell cytotoxicity.

scholarly journals Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

2001 ◽  
Vol 183 (17) ◽  
pp. 5187-5197 ◽  
Author(s):  
Vanessa Sperandio ◽  
Alfredo G. Torres ◽  
Jorge A. Girón ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Mechanism ◽  
Sos Response ◽  
Enterohemorrhagic Escherichia Coli ◽  
Trna Genes ◽  
Wild Type ◽  
E Coli

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenicluxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in theluxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, theluxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

scholarly journals Activation of 2,4-Diaminoquinazoline in Mycobacterium tuberculosis by Rv3161c, a Putative Dioxygenase

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Eduard Melief ◽  
Shilah A. Bonnett ◽  
Edison S. Zuniga ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type A ◽  
Wild Type ◽  
Metabolite Analysis ◽  
Content Type ◽  
Data Support ◽  
In The Wild

ABSTRACT The diaminoquinazoline series has good potency against Mycobacterium tuberculosis. Resistant isolates have mutations in Rv3161c, a putative dioxygenase. We carried out metabolite analysis on a wild-type strain and an Rv3161c mutant strain after exposure to a diaminoquinazoline. The parental compound was found in intracellular extracts from the mutant but not the wild type. A metabolite consistent with a monohydroxylated form was identified in the wild type. These data support the hypothesis that Rv3161c metabolizes diaminoquinazolines in M. tuberculosis.

scholarly journals Sphingolipid C-9 Methyltransferases Are Important for Growth and Virulence but Not for Sensitivity to Antifungal Plant Defensins in Fusarium graminearum

2008 ◽  
Vol 8 (2) ◽  
pp. 217-229 ◽  
Author(s):  
Vellaisamy Ramamoorthy ◽  
Edgar B. Cahoon ◽  
Mercy Thokala ◽  
Jagdeep Kaur ◽  
Jia Li ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Type Strain ◽  
Wild Type Strain ◽  
Fungal Growth ◽  
Wild Type ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Disease Symptoms ◽  
Plant Defensins ◽  
Antifungal Action

ABSTRACT The C-9-methylated glucosylceramides (GlcCers) are sphingolipids unique to fungi. They play important roles in fungal growth and pathogenesis, and they act as receptors for some antifungal plant defensins. We have identified two genes, FgMT1 and FgMT2, that each encode a putative sphingolipid C-9 methyltransferase (C-9-MT) in the fungal pathogen Fusarium graminearum and complement a Pichia pastoris C-9-MT-null mutant. The ΔFgmt1 mutant produced C-9-methylated GlcCer like the wild-type strain, PH-1, whereas the ΔFgmt2 mutant produced 65 to 75% nonmethylated and 25 to 35% methylated GlcCer. No ΔFgmt1ΔFgmt2 double-knockout mutant producing only nonmethylated GlcCer could be recovered, suggesting that perhaps C-9-MTs are essential in this pathogen. This is in contrast to the nonessential nature of this enzyme in the unicellular fungus P. pastoris. The ΔFgmt2 mutant exhibited severe growth defects and produced abnormal conidia, while the ΔFgmt1 mutant grew like the wild-type strain, PH-1, under the conditions tested. The ΔFgmt2 mutant also exhibited drastically reduced disease symptoms in wheat and much-delayed disease symptoms in Arabidopsis thaliana. Surprisingly, the ΔFgmt2 mutant was less virulent on different host plants tested than the previously characterized ΔFggcs1 mutant, which lacks GlcCer synthase activity and produces no GlcCer at all. Moreover, the ΔFgmt1 and ΔFgmt2 mutants, as well as the P. pastoris strain in which the C-9-MT gene was deleted, retained sensitivity to the antifungal plant defensins MsDef1 and RsAFP2, indicating that the C-9 methyl group is not a critical structural feature of the GlcCer receptor required for the antifungal action of plant defensins.

scholarly journals Mechanism of PQQ and CoQ10 overproduction in Methylobacterium as revealed by comparative genomic analysis between mutant and wild-type strains

2020 ◽  
Author(s):  
Changle Zhao ◽  
Yinping Wan ◽  
Xiaojie Cao ◽  
Huili Zhang ◽  
Xin Bao
Keyword(s):  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Genomic Analysis ◽  
Comparative Genomic Analysis ◽  
Comparative Genomic ◽  
Regulation Mechanism ◽  
Whole Genome ◽  
Wild Type ◽  
Coq10 Production

Abstract Background The microbial synthesis of pyrroloquinoline quinone (PQQ) and Coenzyme Q10 (CoQ10) remains the most promising industrial production route. Methylobacterium has been used to generate PQQ and other value-added chemicals from cheap carbon feedstocks.However, the low PQQ and CoQ10 production capacity of the Methylobacterium strains is a major limitation The regulation mechanism for PQQ and CoQ10 biosynthesis in this strain has also not been fully elucidated. Results Methylobacterium sp. CLZ strain was isolated from soil contaminated with chemical wastewater, which can simultaneously produce PQQ, CoQ10, and carotenoids by using cheap methanol as carbon source. We investigated a mutant strain NI91, which increased the PQQ and CoQ10 yield by 72.44% and 59.80%, respectively. Whole-genome sequencing of NI91 and wild-type strain CLZ revealed that both contain a 5.28 Mb chromosome. The comparative genomic analysis and validation study revealed that a significant increase in biomass and PQQ production was associated with the base mutations in the methanol dehydrogenase (MDH) synthesis genes, mxaD and mxaJ. The significant increase in CoQ10 production may be associated with the base mutations in dxs gene, a key gene in the MEP/DOXP pathway. Conclusions A PQQ producing strain that simultaneously produces CoQ10 and carotenoids was selected and after ANI analysis, named as Methylobacterium sp. CLZ. After random mutagenesis of this strain, we obtained NI91 strain, which showed increased production of PQQ and CoQ10. Based on comparative genomic analysis of the whole genome of mutant strain NI91 and wild-type strain CLZ, a total of 270 SNPs and InDels events were detected, which provided a reference for subsequent research. The mutations in mxaD, mxaJ and dxs genes may be related to the high yield of PQQ and CoQ10. These findings will enhance our understanding of the PQQ and CoQ10 over-production mechanism in Methylobacterium sp. NI91 at the genomic level. It will also provide useful clues for strain engineering in order to improve the PQQ and CoQ10 production.

scholarly journals Mutations Conferring Aminoglycoside and Spectinomycin Resistance in Borrelia burgdorferi

2006 ◽  
Vol 50 (2) ◽  
pp. 445-452 ◽  
Author(s):  
Daniel Criswell ◽  
Virginia L. Tobiason ◽  
J. Stephen Lodmell ◽  
D. Scott Samuels
Keyword(s):  
16S Rrna ◽  
Borrelia Burgdorferi ◽  
Type Strain ◽  
Wild Type Strain ◽  
Small Subunit ◽  
Wild Type ◽  
Spectinomycin Resistance ◽  
E Coli ◽  
Resistant Mutants

ABSTRACT We have isolated and characterized in vitro mutants of the Lyme disease agent Borrelia burgdorferi that are resistant to spectinomycin, kanamycin, gentamicin, or streptomycin, antibiotics that target the small subunit of the ribosome. 16S rRNA mutations A1185G and C1186U, homologous to Escherichia coli nucleotides A1191 and C1192, conferred >2,200-fold and 1,300-fold resistance to spectinomycin, respectively. A 16S rRNA A1402G mutation, homologous to E. coli A1408, conferred >90-fold resistance to kanamycin and >240-fold resistance to gentamicin. Two mutations were identified in the gene for ribosomal protein S12, at a site homologous to E. coli residue Lys-87, in mutants selected in streptomycin. Substitutions at codon 88, K88R and K88E, conferred 7-fold resistance and 10-fold resistance, respectively, to streptomycin on B. burgdorferi. The 16S rRNA A1185G and C1186U mutations, associated with spectinomycin resistance, appeared in a population of B. burgdorferi parental strain B31 at a high frequency of 6 × 10−6. These spectinomycin-resistant mutants successfully competed with the wild-type strain during 100 generations of coculture in vitro. The aminoglycoside-resistant mutants appeared at a frequency of 3 × 10−9 to 1 ×10−7 in a population and were unable to compete with wild-type strain B31 after 100 generations. This is the first description of mutations in the B. burgdorferi ribosome that confer resistance to antibiotics. These results have implications for the evolution of antibiotic resistance, because the 16S rRNA mutations conferring spectinomycin resistance have no significant fitness cost in vitro, and for the development of new selectable markers.

scholarly journals A Natural Mutation Involving both Pathogenicity and Perithecium Formation in the Fusarium graminearum Species Complex

2016 ◽  
Vol 6 (12) ◽  
pp. 3883-3892 ◽  
Author(s):  
Haruhisha Suga ◽  
Koji Kageyama ◽  
Masafumi Shimizu ◽  
Misturo Hyakumachi
Keyword(s):  
Mutant Strain ◽  
Fusarium Graminearum ◽  
Type Strain ◽  
Species Complex ◽  
Wild Type Strain ◽  
Genetic Locus ◽  
Chromosome 2 ◽  
Wild Type ◽  
Head Blight ◽  
Fusarium Graminearum Species Complex

Abstract Members of the Fusarium graminearum species complex (Fg complex or FGSC) are the primary pathogens causing Fusarium head blight in wheat and barley worldwide. A natural pathogenicity mutant (strain 0225022) was found in a sample of the Fg complex collected in Japan. The mutant strain did not induce symptoms in wheat spikes beyond the point of inoculation, and did not form perithecia. No segregation of phenotypic deficiencies occurred in the progenies of a cross between the mutant and a fully pathogenic wild-type strain, which suggested that a single genetic locus controlled both traits. The locus was mapped to chromosome 2 by using sequence-tagged markers; and a deletion of ∼3 kb was detected in the mapped region of the mutant strain. The wild-type strain contains the FGSG_02810 gene, encoding a putative glycosylphosphatidylinositol anchor protein, in this region. The contribution of FGSG_02810 to pathogenicity and perithecium formation was confirmed by complementation in the mutant strain using gene transfer, and by gene disruption in the wild-type strain.

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