Evidence for plasmid-mediated chemotaxis ofPseudomonas putidatowards naphthalene and salicylate

A naphthalene (Nap) and salicylate (Sal) degrading microorganism, Pseudomonas putida RKJ1, is chemotactic towards these compounds. This strain carries a 83 kb plasmid. A 25 kb EcoRI fragment of the plasmid contains the genes responsible for Nap degradation through Sal. RKJ5, the plasmid-cured derivative of RKJ1, is neither capable of degradation nor is chemotactic towards Nap or Sal. The recombinant plasmid pRKJ3, which contained a 25 kb EcoRI fragment, was transferred back into the plasmid-free wild-type strain RKJ5, and the transconjugant showed both degradation and chemotaxis. The recombinant plasmid pRKJ3 was also transferred into motile, plasmid-free P. putida KT2442. The resulting transconjugant (RKJ15) showed chemotaxis towards both Nap and Sal. Two mutant strains carrying deletions in pRKJ3 (in KT2442) with phenotypes Nap-Sal+and Nap-Sal-, were also tested for chemotaxis. It was found that the Nap-Sal+mutant strain showed chemotaxis towards Sal only, whereas the Nap-Sal-mutant strain is non-chemotactic towards both the compounds. These results suggest that the metabolism of Nap and Sal may be required for the chemotactic activity.Key words: Pseudomonas putida, plasmid-encoded chemotaxis, naphthalene, salicylate.

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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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Identification and Testing of Porphyromonas gingivalis Virulence Genes with a pPGIVET System

Infection and Immunity ◽

10.1128/iai.70.2.928-937.2002 ◽

2002 ◽

Vol 70 (2) ◽

pp. 928-937 ◽

Cited By ~ 22

Author(s):

Yi Wu ◽

Seok-Woo Lee ◽

Jeffrey D. Hillman ◽

Ann Progulske-Fox

Keyword(s):

Mutant Strain ◽

Porphyromonas Gingivalis ◽

Type Strain ◽

Virulence Genes ◽

Wild Type Strain ◽

Receptor Protein ◽

Wild Type ◽

Kb Cells ◽

Mutant Strains

ABSTRACT An in vivo expression technology (IVET) system was designed to identify previously unknown virulence genes of Porphyromonas gingivalis. Fourteen ivi (for in vivo induced) genes that are induced during infection in a mouse abscess model were identified in our study. Of these, seven had homology to genes in the NCBI database, and the rest had no homology to reported DNA sequences. In order to determine virulence-related properties of these genes, three mutant strains, deleted of ivi8 (no homology to genes in the database), ivi10 (homologous to a putative TonB-dependent outer membrane receptor protein), and ivi11 (an immunoreactive 33-kDa antigen PG125 in P. gingivalis), were created. The mutants were tested in a mouse abscess model for alterations in virulence relative to the wild type by a competition assay in BALB/c mice. After 5 days we observed the enrichment of the wild-type strain over mutant strains Δivi10 and Δivi11, which indicated that mutant strains Δivi10 and Δivi11 are less able to survive in this model than the wild-type strain, while Δivi8 survives as well as the wild-type strain. We propose that knockout of these ivi genes reduced the ability of the mutated P. gingivalis to survive and cause infection compared to the wild-type strain at the site of injection. Also, in separate experiments, groups of mice were challenged with subcutaneous injections of each individual mutant strain (Δivi8, Δivi10, and Δivi11) or with the wild-type strain alone and were then examined to assess their general health status. The results showed that knockout of these ivi genes conferred a reduction in virulence. The ability of the mutants to invade KB cells compared to the wild type was also determined. Interestingly, the CFU counts of the mutant strain Δivi10 recovered from KB cells were eight times lower than those of the wild type, indicating that this mutant has a lower capacity for invasion. These results demonstrate that IVET is a powerful tool in discovering virulence genes and the significant role that ivi genes play in the pathogenesis of this species.

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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

Journal of Bacteriology ◽

10.1128/jb.183.14.4127-4133.2001 ◽

2001 ◽

Vol 183 (14) ◽

pp. 4127-4133 ◽

Cited By ~ 25

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.

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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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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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Cloning, Sequencing, and Characterization of the SdeAB Multidrug Efflux Pump of Serratia marcescens

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.4.1495-1501.2005 ◽

2005 ◽

Vol 49 (4) ◽

pp. 1495-1501 ◽

Cited By ~ 31

Author(s):

Ayush Kumar ◽

Elizabeth A. Worobec

Keyword(s):

Serratia Marcescens ◽

Type Strain ◽

Wild Type Strain ◽

Genomic Library ◽

Efflux Pump ◽

Sodium Dodecyl ◽

Wild Type ◽

Diverse Range ◽

Mutant Strains ◽

Encoding Genes

ABSTRACT Serratia marcescens is an important nosocomial agent known for causing various infections in immunocompromised individuals. Resistance of this organism to a broad spectrum of antibiotics makes the treatment of infections very difficult. This study was undertaken to identify multidrug resistance efflux pumps in S. marcescens. Three mutant strains of S. marcescens were isolated in vitro by the serial passaging of a wild-type strain in culture medium supplemented with ciprofloxacin, norfloxacin, or ofloxacin. Fluoroquinolone accumulation assays were performed to detect the presence of a proton gradient-dependent efflux mechanism. Two of the mutant strains were found to be effluxing norfloxacin, ciprofloxacin, and ofloxacin, while the third was found to efflux only ofloxacin. A genomic library of S. marcescens wild-type strain UOC-67 was constructed and screened for RND pump-encoding genes by using DNA probes for two putative RND pump-encoding genes. Two different loci were identified: sdeAB, encoding an MFP and an RND pump, and sdeCDE, encoding an MFP and two different RND pumps. Northern blot analysis revealed overexpression of sdeB in two mutant strains effluxing fluoroquinolones. Analysis of the sdeAB and sdeCDE loci in Escherichia coli strain AG102MB, deficient in the RND pump (AcrB), revealed that gene products of sdeAB are responsible for the efflux of a diverse range of substrates that includes ciprofloxacin, norfloxacin, ofloxacin, chloramphenicol, sodium dodecyl sulfate, ethidium bromide, and n-hexane, while those of sdeCDE did not result in any change in susceptibilities to any of these agents.

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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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Inactivation of the Crp/Fnr Family of Regulatory Genes in Listeria monocytogenes Strain F2365 Does Not Alter Its Heat Resistance at 60°C†

Journal of Food Protection ◽

10.4315/0362-028x-69.11.2758 ◽

2006 ◽

Vol 69 (11) ◽

pp. 2758-2760 ◽

Cited By ~ 2

Author(s):

DARRELL O. BAYLES ◽

GAYLEN A. UHLICH

Keyword(s):

Listeria Monocytogenes ◽

Heat Resistance ◽

Thermal Tolerance ◽

Type Strain ◽

Transcriptional Control ◽

Wild Type Strain ◽

Gene Cassette ◽

Wild Type ◽

Mutant Strains ◽

Virulence Regulator

A surprising facet of the Listeria monocytogenes genome is the presence of 15 genes that code for regulators in the Crp/Fnr family and include the virulence regulator PrfA. The genes under the transcriptional control of these regulators are currently undetermined, with the exception of some genes controlled by the major virulence regulator PrfA. Using 12 strains of L. monocytogenes, each with an inserted gene cassette that interrupts and renders nonfunctional a different L. monocytogenes strain F2365 Crp/Fnr regulator, we heat challenged each strain at 60°C with an immersed-coil heating apparatus, modeled the survivor data to calculate the underlying mean and mode of the heat resistance distribution for each strain, and compared the thermal tolerance of each mutant to the wild-type strain to determine if any of the Crp/Fnr mutants demonstrated altered heat tolerance. All 12 of the Crp/Fnr mutant strains tested had heat resistance characteristics similar to the wild-type strain (P > 0.05), indicating that mutations in these Crp/Fnr genes neither increased nor decreased the sensitivity of L. monocytogenes strain F2365 to mild heat.

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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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