The Rhizobium etli metZ Gene Is Essential for Methionine Biosynthesis and Nodulation of Phaseolus vulgaris

A mutant strain (CTNUX23) of Rhizobium etli carrying Tn5 unable to grow with sulfate as the sole sulfur source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a metZ (O-succinylhomoserine sulfhydrylase)-homologous gene. The CTNUX23 mutant strain had a growth dependency for methionine, although cystathionine or homocysteine, but not homoserine or O-succinylhomoserine, allowed growth of the mutant. RNase protection assays showed that the metZ-like gene had a basal level of expression in methionine- or cysteine-grown cells, which was induced when sulfate or thiosulfate was used. The metZ gene was cloned from the parent wild-type strain, CE3, and the resulting plasmid pAR204 relieved, after transformation, the methionine auxotrophy of both strains CTNUX23 of R. etli and PAO503(metZ) of Pseudomonas aeruginosa. Unlike strain CE3 or CTNUX23 (pAR204), strain CTNUX23 showed undetectable levels of O-succinylhomoserine sulfhydrylase activity. Strain CTNUX23 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) or to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris, unless methionine was added to the growth medium. These data and our previous results support the notion that cysteine or glutathione, but not methionine, is supplied by the root cells to bacteria growing inside the plant.

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The Rhizobium etli argC Gene Is Essential for Arginine Biosynthesis and Nodulation of Phaseolus vulgaris

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.2.250 ◽

2001 ◽

Vol 14 (2) ◽

pp. 250-254 ◽

Cited By ~ 11

Author(s):

Simona Ferraioli ◽

Rosarita Taté ◽

Emilia Caputo ◽

Alessandro Lamberti ◽

Anna Riccio ◽

...

Keyword(s):

Phaseolus Vulgaris ◽

Mutant Strain ◽

Wild Type Strain ◽

Reductase Activity ◽

Homologous Gene ◽

Rhizobium Etli ◽

Wild Type ◽

Arginine Biosynthesis ◽

Nodulation Factors ◽

Wild Type Parent

A Tn5-induced mutant strain (CTNUX5) of Rhizobium etli unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into an argC-homologous gene. Unlike its wild-type parent (strain CE3), the mutant strain CTNUX5 had an absolute dependency on arginine to grow. The argC gene was cloned from the wild-type strain CE3, and the resulting plasmid, pAR207, after transformation was shown to relieve the arginine auxotrophy of strain CTNUX5. Unlike strain CE3 or CTNUX5-pAR207, strain CTNUX5 showed undetectable levels of N-acetyl-γ-glutamylphosphate reductase activity. Unless ar-ginine was added to the growth medium, strain CTNUX5 was unable to produce flavonoid-inducible lipo-chitin oli-gosaccharides (nodulation factors) and to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris.

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The Rhizobium etli trpB Gene Is Essential for an Effective Symbiotic Interaction with Phaseolus vulgaris

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1999.12.10.926 ◽

1999 ◽

Vol 12 (10) ◽

pp. 926-933 ◽

Cited By ~ 12

Author(s):

Rosarita Taté ◽

Anna Riccio ◽

Emilia Caputo ◽

Michele Cermola ◽

Renee Favre ◽

...

Keyword(s):

Phaseolus Vulgaris ◽

Homologous Gene ◽

Tryptophan Synthase ◽

Sole Nitrogen Source ◽

Tryptophan Biosynthesis ◽

Rhizobium Etli ◽

Nod Factors ◽

Free Living ◽

Symbiotic Interaction ◽

Free Living Conditions

A mutant strain (CTNUX4) of Rhizobium etli carrying Tn5 unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a trpB (tryptophan synthase)-homologous gene. When tested on the roots of Phaseolus vulgaris, strain CTNUX4 was able to induce only small, slightly pink, ineffective (Fix¯) nodules. However, under free-living conditions, strain CTNUX4 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) unless tryptophan was added to the growth medium. These data and histological observations indicate that the lack of tryptophan biosynthesis affects the symbiotic behavior of R. etli.

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

BMC Microbiology ◽

10.1186/s12866-020-02083-0 ◽

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.

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Establishing Virulence Associated Polyphosphate Kinase 2 as a drug target for Mycobacterium tuberculosis

Scientific Reports ◽

10.1038/srep26900 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 13

Author(s):

Mamta Singh ◽

Prabhakar Tiwari ◽

Garima Arora ◽

Sakshi Agarwal ◽

Saqib Kidwai ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Mutant Strain ◽

Guinea Pigs ◽

High Throughput Screening ◽

Drug Target ◽

Wild Type Strain ◽

Wild Type ◽

Inorganic Polyphosphate ◽

Phosphate Donor ◽

Microbial Stress

Abstract Inorganic polyphosphate (PolyP) plays an essential role in microbial stress adaptation, virulence and drug tolerance. The genome of Mycobacterium tuberculosis encodes for two polyphosphate kinases (PPK-1, Rv2984 and PPK-2, Rv3232c) and polyphosphatases (ppx-1, Rv0496 and ppx-2, Rv1026) for maintenance of intracellular PolyP levels. Microbial polyphosphate kinases constitute a molecular mechanism, whereby microorganisms utilize PolyP as phosphate donor for synthesis of ATP. In the present study we have constructed ppk-2 mutant strain of M. tuberculosis and demonstrate that PPK-2 enzyme contributes to its ability to cause disease in guinea pigs. We observed that ppk-2 mutant strain infected guinea pigs had significantly reduced bacterial loads and tissue pathology in comparison to wild type infected guinea pigs at later stages of infection. We also report that in comparison to the wild type strain, ppk-2 mutant strain was more tolerant to isoniazid and impaired for survival in THP-1 macrophages. In the present study we have standardized a luciferase based assay system to identify chemical scaffolds that are non-cytotoxic and inhibit M. tuberculosis PPK-2 enzyme. To the best of our knowledge this is the first study demonstrating feasibility of high throughput screening to obtain small molecule PPK-2 inhibitors.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01505-18 ◽

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.

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

10.21203/rs.3.rs-36145/v1 ◽

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.

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

G3 Genes|Genome|Genetics ◽

10.1534/g3.116.033951 ◽

2016 ◽

Vol 6 (12) ◽

pp. 3883-3892 ◽

Cited By ~ 3

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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Growth Inhibition of the Ralstonia solanacearum Wild-type Strain in a Culture Filtrate of Phenotypic Conversion Mutant Strain

Environment Control in Biology ◽

10.2525/ecb.54.133 ◽

2016 ◽

Vol 54 (3) ◽

pp. 133-138 ◽

Cited By ~ 2

Author(s):

Hiroki NAKAHARA ◽

Taro MORI ◽

Hiromi MATSUSAKI ◽

Naotaka MATSUZOE

Keyword(s):

Growth Inhibition ◽

Mutant Strain ◽

Culture Filtrate ◽

Ralstonia Solanacearum ◽

Type Strain ◽

Wild Type Strain ◽

Wild Type

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Identification of a Third Sulfate Activation System in Sinorhizobium sp. Strain BR816: the CysDN Sulfate Activation Complex

Applied and Environmental Microbiology ◽

10.1128/aem.69.4.2006-2014.2003 ◽

2003 ◽

Vol 69 (4) ◽

pp. 2006-2014 ◽

Cited By ~ 18

Author(s):

Carla Snoeck ◽

Christel Verreth ◽

Ismael Hernández-Lucas ◽

Esperanza Martínez-Romero ◽

Jos Vanderleyden

Keyword(s):

Growth Characteristics ◽

Open Reading Frames ◽

Lag Phase ◽

Sulfur Source ◽

Nod Factors ◽

Wild Type ◽

Triple Mutant ◽

Nod Factor ◽

Growth Defects ◽

Sulfate Activation

ABSTRACT Sinorhizobium sp. strain BR816 possesses two nodPQ copies, providing activated sulfate (3′-phosphoadenosine-5′-phosphosulfate [PAPS]) needed for the biosynthesis of sulfated Nod factors. It was previously shown that the Nod factors synthesized by a nodPQ double mutant are not structurally different from those of the wild-type strain. In this study, we describe the characterization of a third sulfate activation locus. Two open reading frames were fully characterized and displayed the highest similarity with the Sinorhizobium meliloti housekeeping ATP sulfurylase subunits, encoded by the cysDN genes. The growth characteristics as well as the levels of Nod factor sulfation of a cysD mutant (FAJ1600) and a nodP1 nodQ2 cysD triple mutant (FAJ1604) were determined. FAJ1600 shows a prolonged lag phase only with inorganic sulfate as the sole sulfur source, compared to the wild-type parent. On the other hand, FAJ1604 requires cysteine for growth and produces sulfate-free Nod factors. Apigenin-induced nod gene expression for Nod factor synthesis does not influence the growth characteristics of any of the strains studied in the presence of different sulfur sources. In this way, it could be demonstrated that the “household” CysDN sulfate activation complex of Sinorhizobium sp. strain BR816 can additionally ensure Nod factor sulfation, whereas the symbiotic PAPS pool, generated by the nodPQ sulfate activation loci, can be engaged for sulfation of amino acids. Finally, our results show that rhizobial growth defects are likely the reason for a decreased nitrogen fixation capacity of bean plants inoculated with cysD mutant strains, which can be restored by adding methionine to the plant nutrient solution.

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Peptides’ involvement in Pseudomonas putida biofilm formation

10.5194/biofilms9-38 ◽

2020 ◽

Author(s):

Riho Teras ◽

Hanna Ainelo ◽

Marge Puhm

Keyword(s):

Pseudomonas Putida ◽

Mutant Strain ◽

Type Strain ◽

Deletion Mutant ◽

Wild Type Strain ◽

Positive Impact ◽

Proteinase K ◽

Wild Type ◽

Positive Effect ◽

Positively Charged

Pseudomonas putida rapidly forms a biofilm, after which its biomass usually disperses to half its initial amount. We have observed different biofilm dynamics of P. putida in a complex medium LB and a minimal medium M9+glc+CAA and inquired about the importance of extracellular factors for the formation of P. putida biofilm. The proteinaceous component of LB increases the biomass of P. putida biofilm. Supplementation of M9 with tryptone but not CAA increased the biofilm biomass. Proteinase K treatment of LB medium reduced the biomass of P. putida biofilm. At the same time, growth rate or maximum OD of planktic bacteria in used media did not correlate with biofilm biomass of the same media. Thus, peptides appeared to have a positive effect on the biofilm as an extracellular factor and not as a source of C and N. We replaced tryptone in M9 medium with positively charged poly-L-lysine (MW. 1000-5000 Da), negatively charged poly-L-glutaminic acid (MW. 1500-5500 Da) or neutral poly-LD-alanine (MW. 3000-7000). Poly-lysine and poly-glutamic acid had a slight positive effect on the biomass of P. putida wild type strain PSm biofilm and poly-alanine did not affect the biofilm. We have previously shown that overexpression of fis in P. putida strain F15 increases biofilm biomass by increasing the lapA expression, the main adhesin gene of biofilm. Using media similar to that used for the wild-type strain for strain F15, we ascertained that only poly-lysine out of these three polypeptides restored the positive effect of fis-overexpression on the biofilm biomass. At the same time, the positive impact of fis-overexpression was absent in lapA deletion mutant strain, but not in lapF deletion mutant strain. In conclusion, the formation of P. putida biofilm depends on polypeptides in the environment. The enhancing effect of positively charged polypeptides appears to be evident in the presence of LapA, a key factor for P. putida biofilm.

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