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

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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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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Amylase hyper-producing haploid recombinant strains of Thermomyces lanuginosus obtained by intraspecific protoplast fusion

Canadian Journal of Microbiology ◽

10.1139/w00-042 ◽

2000 ◽

Vol 46 (7) ◽

pp. 669-673 ◽

Cited By ~ 3

Author(s):

K Rubinder ◽

B S Chadha ◽

S Singh ◽

H S Saini

Keyword(s):

Protoplast Fusion ◽

Culture Filtrate ◽

Catabolite Repression ◽

Type Strain ◽

Wild Type Strain ◽

Thermomyces Lanuginosus ◽

Wild Type ◽

Haploid Strain ◽

Recombinant Strains ◽

Specific Activities

Amylase hyper-producing, catabolite-repression-resistant, recombinant strains were produced by intraspecific protoplast fusion of thermophilic fungus Thermomyces lanuginosus strains, using well-characterized, morphological, and 2-deoxy-D-glucose resistant markers. The fusant heterokaryons exhibited enhanced amylase activities as compared to the amylase hyper-producing parental strain (T2). Diploids derived from heterokaryons segregated to stable haploid recombinant strains. In the haploid strain (Tlh 4q), approximately 5-fold higher specific activities of α-amylase and glucoamylase in the culture filtrate were observed as compared to the wild-type strain (W0).Key words: Thermomyces lanuginosus, protoplast fusion, amylase hyper-producing strain, catabolite repression.

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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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Competition with the Wild-type Strain and a Phenotypic Conversion Strain of Ralstonia solanacearum in the Soil and Plants

Shokubutsu Kankyo Kogaku ◽

10.2525/shita.28.133 ◽

2016 ◽

Vol 28 (3) ◽

pp. 133-141 ◽

Cited By ~ 2

Author(s):

Hiroki NAKAHARA ◽

Taro MORI ◽

Hiromi MATSUSAKI ◽

Naotaka MATSUZOE

Keyword(s):

Ralstonia Solanacearum ◽

Type Strain ◽

Wild Type Strain ◽

Wild Type

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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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PhoU enhances the ability of extraintestinal pathogenic Escherichia coli strain CFT073 to colonize the murine urinary tract

Microbiology ◽

10.1099/mic.0.28281-0 ◽

2006 ◽

Vol 152 (1) ◽

pp. 153-160 ◽

Cited By ~ 23

Author(s):

Eric L. Buckles ◽

Xiaolin Wang ◽

C. Virginia Lockatell ◽

David E. Johnson ◽

Michael S. Donnenberg

Keyword(s):

Escherichia Coli ◽

Urinary Tract Infection ◽

Urinary Tract ◽

Tract Infection ◽

Mutant Strain ◽

Human Urine ◽

Type Strain ◽

Wild Type Strain ◽

Wild Type ◽

Strain Cft073

The phoU gene is the last cistron in the pstSCAB–phoU operon and functions as a negative regulator of the Pho regulon. The authors previously identified a phoU mutant of extraintestinal pathogenic Escherichia coli strain CFT073 and demonstrated that this mutant was attenuated for survival in the murine model of ascending urinary tract infection. It is hypothesized that the PhoU protein might serve as a urovirulence factor by indirectly affecting the expression of virulence-related genes. In this study, the phoU mutant was further characterized and PhoU was confirmed as a virulence factor. Western blot analysis demonstrated that insertion of the transposon in the phoU gene disrupted the expression of PhoU. The phoU mutant had derepressed alkaline phosphatase activity under phosphate-excess and -limiting conditions. In single-challenge murine ascending urinary tract infection experiments, quantitative cultures of urine, bladder and kidney revealed no significant differences between the phoU mutant strain and the wild-type strain CFT073. However, in competitive colonization experiments, the phoU mutant strain was significantly out-competed by the wild-type strain in the kidneys and urine and recovered in lower amount in the bladder. Complementation of the phoU mutant with a plasmid containing the wild-type phoU gene restored the expression of PhoU and alkaline phosphate activity to wild-type levels and no significant difference in colonization was observed between the phoU mutant containing the complementing plasmid and wild-type in competitive colonization experiments. In human urine, the phoU mutant and wild-type grew comparably when inoculated independently, indicating that the attenuation observed was not due to a general growth defect. However, as observed in vivo, the wild-type out-competed the phoU mutant in competition growth experiments in human urine. These data indicate that PhoU contributes to efficient colonization of the murine urinary tract and add PhoU to a short list of confirmed urovirulence factors.

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Glutathione Reductase from Lactobacillus sanfranciscensis DSM20451T: Contribution to Oxygen Tolerance and Thiol Exchange Reactions in Wheat Sourdoughs

Applied and Environmental Microbiology ◽

10.1128/aem.02322-06 ◽

2007 ◽

Vol 73 (14) ◽

pp. 4469-4476 ◽

Cited By ~ 57

Author(s):

André Jänsch ◽

Maher Korakli ◽

Rudi F. Vogel ◽

Michael G. Gänzle

Keyword(s):

Growth Rate ◽

Glutathione Reductase ◽

Mutant Strain ◽

Type Strain ◽

Wild Type Strain ◽

Wild Type ◽

Exchange Reactions ◽

Aerobic Growth ◽

Oxygen Tolerance ◽

Lactobacillus Sanfranciscensis

ABSTRACT The effect of the glutathione reductase (GshR) activity of Lactobacillus sanfranciscensis DSM20451T on the thiol levels in fermented sourdoughs was determined, and the oxygen tolerance of the strain was also determined. The gshR gene coding for a putative GshR was sequenced and inactivated by single-crossover integration to yield strain L. sanfranciscensis DSM20451TΔgshR. The gene disruption was verified by sequencing the truncated gshR and surrounding regions on the chromosome. The gshR activity of L. sanfranciscensis DSM20451TΔgshR was strongly reduced compared to that of the wild-type strain, demonstrating that gshR indeed encodes an active GshR enzyme. The thiol levels in wheat doughs fermented with L. sanfranciscensis DSM20451 increased from 9 μM to 10.5 μM sulfhydryl/g of dough during a 24-h sourdough fermentation, but in sourdoughs fermented with L. sanfranciscensis DSM20451TΔgshR and in chemically acidified doughs, the thiol levels decreased to 6.5 to 6.8 μM sulfhydryl/g of dough. Remarkably, the GshR-negative strains Lactobacillus pontis LTH2587 and Lactobacillus reuteri BR11 exerted effects on thiol levels in dough comparable to those of L. sanfranciscensis. In addition to the effect on thiol levels in sourdough, the loss of GshR activity in L. sanfranciscensis DSM20451TΔgshR resulted in a loss of oxygen tolerance. The gshR mutant strain exhibited a strongly decreased aerobic growth rate on modified MRS medium compared to either the growth rate under anaerobic conditions or that of the wild-type strain, and aerobic growth was restored by the addition of cysteine. Moreover, the gshR mutant strain was more sensitive to the superoxide-generating agent paraquat.

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Involvement of the XpsN Protein in Formation of the XpsL-XpsM Complex in Xanthomonas campestris pv. campestris Type II Secretion Apparatus

Journal of Bacteriology ◽

10.1128/jb.183.2.528-535.2001 ◽

2001 ◽

Vol 183 (2) ◽

pp. 528-535 ◽

Cited By ~ 16

Author(s):

Hsien-Ming Lee ◽

Shiaw-Wei Tyan ◽

Wei-Ming Leu ◽

Ling-Yun Chen ◽

David Chanhen Chen ◽

...

Keyword(s):

Gene Cluster ◽

Mutant Strain ◽

Type Strain ◽

Xanthomonas Campestris ◽

Wild Type Strain ◽

Type Ii ◽

Wild Type ◽

In The Wild ◽

Steady State Levels ◽

Type Gene

ABSTRACT The xps gene cluster is required for the second step of type II protein secretion in Xanthomonas campestrispv. campestris. Deletion of the entire gene cluster caused accumulation of secreted proteins in the periplasm. By analyzing protein abundance in the chromosomal mutant strains, we observed mutual dependence for normal steady-state levels between the XpsL and the XpsM proteins. The XpsL protein was undetectable in total lysate prepared from thexpsM mutant strain, and vice versa. Introduction of the wild-type xpsM gene carried on a plasmid into thexpsM mutant strain was sufficient for reappearance of the XpsL protein, and vice versa. Moreover, both XpsL and XpsM proteins were undetectable in the xpsN mutant strain. They were recovered either by reintroducing the wild-type xpsNgene or by introducing extra copies of wild-type xpsL orxpsM individually. Overproduction of wild-type XpsL and -M proteins simultaneously, but not separately, in the wild-type strain of X. campestris pv. campestris caused inhibition of secretion. Complementation of an xpsL orxpsM mutant strain with a plasmid-borne wild-type gene was inhibited by coexpression of XpsL and XpsM. The presence of the xpsN gene on the plasmid along with thexpsL and the xpsM genes caused more severe inhibition in both cases. Furthermore, complementation of thexpsN mutant strain was also inhibited. In both the wild-type strain and a strain with the xps gene cluster deleted (XC17433), carrying pCPP-LMN, which encodes all three proteins, each protein coprecipitated with the other two upon immunoprecipitation. Expression of pairwise combinations of the three proteins in XC17433 revealed that the XpsL-XpsM and XpsM-XpsN pairs still coprecipitated, whereas the XpsL-XpsN pair no longer coprecipitated.

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