Identification of Klebsiella Variicola T29A Genes Involved In Tolerance To Desiccation

Introduction: Several plant-beneficial bacteria have the capability to promote the growth of plants through different mechanisms. The survival of such bacteria could be affected by environmental abiotic factors compromising their capabilities of phytostimulation. One of the limiting abiotic factors is low water availability. Materials and Methods: In extreme cases, bacterial cells can suffer desiccation, which triggers harmful effects on cells. Bacteria tolerant to desiccation have developed different strategies to cope with these conditions; however, the genes involved in these processes have not been sufficiently explored. Klebsiella variicola T29A is a beneficial bacterial strain that promotes the growth of corn plants and is highly tolerant to desiccation. In the present work, we investigated genes involved in desiccation tolerance. Results & Discussion: As a result, a library of 8974 mutants of this bacterial strain was generated by random mutagenesis with mini-Tn5 transposon, and mutants that lost the capability to tolerate desiccation were selected. We found 14 sensitive mutants; those with the lowest bacterial survival rate contained mini-Tn5 transposon inserted into genes encoding a protein domain related to BetR, putative secretion ATPase and dihydroorotase. The mutant in the betR gene had the lowest survival; therefore, the mutagenized gene was validated using specific amplification and sequencing. Conclusion: Trans complementation with the wild-type gene improved the survival of the mutant under desiccation conditions, showing that this gene is a determinant for the survival of K. variicola T29A under desiccation conditions.

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Pseudomonas aeruginosa Oligoribonuclease Controls Susceptibility to Polymyxin B by Regulating Pel Exopolysaccharide Production

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02072-21 ◽

2022 ◽

Author(s):

Baopeng Yang ◽

Yujun Jiang ◽

Yongxin Jin ◽

Fang Bai ◽

Zhihui Cheng ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Bacterial Resistance ◽

Defense Mechanism ◽

Extracellular Polysaccharide ◽

Opportunistic Pathogen ◽

Polymyxin B ◽

Guanosine Monophosphate ◽

Extracellular Dna ◽

Bacterial Cells ◽

Bacterial Survival

Polymyxins are considered as the last resort antibiotics to treat infections caused by multidrug-resistant Gram negative pathogens. Pseudomonas aeruginosa is an opportunistic pathogen that causes various infections in humans. Proteins involved in lipopolysaccharide modification and maintaining inner and outer membrane integrities have been found to contribute to the bacterial resistance to polymyxins. Oligoribonuclease (Orn) is an exonuclease that regulates the homeostasis of intracellular (3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), thereby regulating the production of extracellular polysaccharide in P. aeruginosa . Previously, we demonstrated that Orn affects the bacterial resistance to fluoroquinolone, β-lactam and aminoglycoside antibiotics. In this study, we found that mutation of orn increased the bacterial survival following polymyxin B treatment in a wild type P. aeruginosa strain PA14. Overexpression of c-di-GMP degradation enzymes in the orn mutant reduced the bacterial survival. By using a fluorescence labeled polymyxin B, we found that mutation of orn increased the bacterial surface bound polymyxin B. Deletion of the Pel synthesis genes or treatment with a Pel hydrolase reduced the surface bound polymyxin B and bacterial survival. We further demonstrated that Pel binds to extracellular DNA (eDNA), which traps polymyxin B and thus protects the bacterial cells. Collectively, our results revealed a novel defense mechanism against polymyxin in P. aeruginosa .

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Diversity of Free-Living and Attached Bacteria in Offshore Western Mediterranean Waters as Depicted by Analysis of Genes Encoding 16S rRNA

Applied and Environmental Microbiology ◽

10.1128/aem.65.2.514-522.1999 ◽

1999 ◽

Vol 65 (2) ◽

pp. 514-522 ◽

Cited By ~ 222

Author(s):

Silvia G. Acinas ◽

Josefa Antón ◽

Francisco Rodríguez-Valera

Keyword(s):

16S Rdna ◽

Restriction Analysis ◽

Western Mediterranean ◽

Bacterial Cells ◽

Free Living ◽

Attached Bacteria ◽

Genes Encoding ◽

Community Fingerprinting ◽

Close Relatives ◽

Alteromonas Macleodii

ABSTRACT In a previous study (S. G. Acinas, F. Rodrı́guez-Valera, and C. Pedrós-Alió, FEMS Microbiol. Ecol. 24:27–40, 1997), community fingerprinting by 16S rDNA restriction analysis applied to Mediterranean offshore waters showed that the free-living pelagic bacterial community was very different from the bacterial cells aggregated or attached to particles of more than about 8 μm. Here we have studied both assemblages at three depths (5, 50, and 400 m) by cloning and sequencing the 16S rDNA obtained from the same samples, and we have also studied the samples by scanning electron microscopy to detect morphology patterns. As expected, the sequences retrieved from the assemblages were very different. The subsample of attached bacteria contained very little diversity, with close relatives of a well-known species of marine bacteria, Alteromonas macleodii, representing the vast majority of the clones at every depth. On the other hand, the free-living assemblage was highly diverse and varied with depth. At 400 m, close relatives of cultivated γProteobacteria predominated, but as shown by other authors, near the surface most clones were related to phylotypes described only by sequence, in which the α Proteobacteria of the SAR11 cluster predominated. The new technique of rDNA internal spacer analysis has been utilized, confirming these results. Clones representative of the A. macleodii cluster have been completely sequenced, producing a picture that fits well with the idea that they could represent a genus with at least two species and with a characteristic depth distribution.

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Evolution and Sequence Diversity of FhuA inSalmonellaandEscherichia

Infection and Immunity ◽

10.1128/iai.00573-18 ◽

2018 ◽

Vol 86 (11) ◽

Cited By ~ 3

Author(s):

Yejun Wang ◽

Xiongbin Chen ◽

Yueming Hu ◽

Guoqiang Zhu ◽

Aaron P. White ◽

...

Keyword(s):

Gene Mutations ◽

Comparative Sequence Analysis ◽

Sequence Diversity ◽

Bacterial Cells ◽

Bacterial Survival ◽

Bacterial Strains ◽

Content Type ◽

High Conservation ◽

High Selection ◽

Clustering Pattern

ABSTRACTThefhuACDBoperon, present in a number ofEnterobacteriaceae, encodes components essential for the uptake of ferric hydroxamate type siderophores. FhuA acts not only as a transporter for physiologically important chelated ferric iron but also as a receptor for various bacteriophages, toxins, and antibiotics, which are pathogenic to bacterial cells. In this research,fhuAgene distribution and sequence diversity were investigated inEnterobacteriaceae, especiallySalmonellaandEscherichia. Comparative sequence analysis resulted in afhuAphylogenetic tree that did not match the expected phylogeny of species or trees of thefhuCDBgenes. ThefhuAsequences showed a unique mosaic clustering pattern. On the other hand, the gene sequences showed high conservation for strains from the same serovar or serotype. In total, six clusters were identified from FhuA proteins inSalmonellaandEscherichia, among which typical peptide fragment variations could be defined. Six fragmental insertions/deletions and two substitution fragments were discovered, for which the combination of polymorphism patterns could well classify the different clusters. Structural modeling demonstrated that all the six featured insertions/deletions and one substitution fragment are located at the apexes of the long loops present as part of the FhuA external pocket. These frequently mutated regions are likely under high selection pressure, with bacterial strains balancing escape from phage infection or toxin/antibiotics attack viafhuAgene mutations while maintaining the siderophore uptake activity essential for bacterial survival. The unusualfhuAclustering suggests that high-frequency exchange offhuAgenes has occurred between enterobacterial strains after distinctive species were established.

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Fast and reliable procedure developed to generate soft rot Pectobacteriaceae (Pectobacterium spp. and Dickeya spp.) Tn5 mutants resistant to bacteriophage infection

European Journal of Plant Pathology ◽

10.1007/s10658-020-02143-1 ◽

2020 ◽

Vol 159 (1) ◽

pp. 227-231

Author(s):

Robert Czajkowski

Keyword(s):

Growth Medium ◽

Aminolevulinic Acid ◽

Soft Rot ◽

Lytic Bacteriophage ◽

Bacterial Cells ◽

Bacteriophage Infection ◽

Genes Encoding ◽

Tn5 Mutants ◽

Bacterial Mutants ◽

Fast Procedure

AbstractA simple and fast procedure has been developed to generate soft rot Pectobacteriaceae (SRP: Pectobacterium spp. and Dickeya spp.) Tn5 mutants in genes encoding receptors used by bacteriophages to interact with their hosts, for the follow-up studies. The procedure is inexpensive and does not require any specialized tools and/or dedicated technical support. The neomycin-resistant SRP Tn5 mutants are generated via conjugation with a transposon donor Escherichia coli ST18 strain (requiring 5-aminolevulinic acid (5-ALA) to survive) carrying pFAJ1819-mini-Tn5-neoR. The conjugation is done on solid medium supplemented with 5-ALA. After conjugation bacterial cells are collected, suspended in liquid bacterial medium, added to the suspension containing lytic bacteriophages and incubated for the additional 30 min with shaking (120 rpm). During this stage, the transposon recipients (Pectobacterium spp. and/or Dickeya spp. Tn5 mutants), susceptible to bacteriophage infection are lysed. Likewise, due to the lack of 5-ALA in the growth medium, E. coli ST18 (transposon donor) cells die at this stage. Finally, after incubation, the bacterial mutants with the Tn5 insertions, resistant to phage infection are selected on solid growth medium supplemented with neomycin. The Tn5 insertion sites are sequenced to acquire knowledge about the Tn5-distrupted genes and their putative function in phage-host interactions. The proposed assay allows generation of a number of immediately-available Tn5 mutants expressing phage-resistant phenotypes in a short time (ca. 48 h) that can be later characterized for various other phenotypic features. In this study, as a proof-of-concept, this method has been used to generate Dickeya solani IPO2222 Tn5 mutants resistant to infection caused by the lytic bacteriophage ɸD5.

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Regulation of tartrate metabolism by TtdR and relation to the DcuS–DcuR-regulated C4-dicarboxylate metabolism of Escherichia coli

Microbiology ◽

10.1099/mic.0.031401-0 ◽

2009 ◽

Vol 155 (11) ◽

pp. 3632-3640 ◽

Cited By ~ 12

Author(s):

Ok Bin Kim ◽

Julia Reimann ◽

Hanna Lukas ◽

Uwe Schumacher ◽

Jan Grimpo ◽

...

Keyword(s):

Escherichia Coli ◽

Promoter Region ◽

Anaerobic Conditions ◽

Two Component System ◽

Two Component ◽

Fumarate Respiration ◽

Mutual Regulation ◽

Genes Encoding ◽

Type Gene ◽

Tartrate Dehydratase

Escherichia coli catabolizes l-tartrate under anaerobic conditions to oxaloacetate by the use of l-tartrate/succinate antiporter TtdT and l-tartrate dehydratase TtdAB. Subsequently, l-malate is channelled into fumarate respiration and degraded to succinate by the use of fumarase FumB and fumarate reductase FrdABCD. The genes encoding the latter pathway (dcuB, fumB and frdABCD) are transcriptionally activated by the DcuS–DcuR two-component system. Expression of the l-tartrate-specific ttdABT operon encoding TtdAB and TtdT was stimulated by the LysR-type gene regulator TtdR in the presence of l- and meso-tartrate, and repressed by O2 and nitrate. Anaerobic expression required a functional fnr gene, and nitrate repression depended on NarL and NarP. Expression of ttdR, encoding TtdR, was repressed by O2, nitrate and glucose, and positively regulated by TtdR and DcuS. Purified TtdR specifically bound to the ttdR–ttdA promoter region. TtdR was also required for full expression of the DcuS–DcuR-dependent dcuB gene in the presence of tartrate. Overall, expression of the ttdABT genes is subject to l-/meso-tartrate-dependent induction, and to aerobic and nitrate repression. The control is exerted directly at ttdA and in addition indirectly by regulating TtdR levels. TtdR recognizes a subgroup (l- and meso-tartrate) of the stimuli perceived by the sensor DcuS, which responds to all C4-dicarboxylates; both systems apparently communicate by mutual regulation of the regulatory genes.

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Amino Acid Transport and Metabolism in Mycobacteria: Cloning, Interruption, and Characterization of anl-Arginine/γ-Aminobutyric Acid Permease inMycobacterium bovis BCG

Journal of Bacteriology ◽

10.1128/jb.182.4.919-927.2000 ◽

2000 ◽

Vol 182 (4) ◽

pp. 919-927 ◽

Cited By ~ 18

Author(s):

Anjali Seth ◽

Nancy D. Connell

Keyword(s):

Amino Acid ◽

Mutant Strain ◽

Nitrogen Sources ◽

Single Copy ◽

Aminobutyric Acid ◽

Cosmid Library ◽

Wild Type ◽

Carbon And Nitrogen ◽

Genes Encoding ◽

Type Gene

ABSTRACT Genes encoding l-arginine biosynthetic and transport proteins have been shown in a number of pathogenic organisms to be important for metabolism within the host. In this study we describe the cloning of a gene (Rv0522) encoding an amino acid transporter fromMycobacterium bovis BCG and the effects of its deletion onl-arginine transport and metabolism. The Rv0522 gene of BCG was cloned from a cosmid library by using primers homologous to therocE gene of Bacillus subtilis, a putative arginine transporter. A deletion mutant strain was constructed by homologous recombination with the Rv0522 gene interrupted by a selectable marker. The mutant strain was complemented with the wild-type gene in single copy. Transport analysis of these strains was conducted using 14C-labeled substrates. Greatly reduced uptake of l-arginine and γ-aminobutyric acid (GABA) but not of lysine, ornithine, proline, or alanine was observed in the mutant strain compared to the wild type, grown in Middlebrook 7H9 medium. However, when the strains were starved for 24 h or incubated in a minimal salts medium containing 20 mM arginine (in which even the parent strain does not grow),l-[14C]arginine uptake by the mutant but not the wild-type strain increased strongly. Exogenousl-arginine but not GABA, lysine, ornithine, or alanine was shown to be toxic at concentrations of 20 mM and above to wild-type cells growing in optimal carbon and nitrogen sources such as glycerol and ammonium. l-Arginine supplied in the form of dipeptides showed no toxicity at concentrations as high as 30 mM. Finally, the permease mutant strain showed no defect in survival in unactivated cultured murine macrophages compared with wild-type BCG.

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Coordinated Zinc Homeostasis Is Essential for the Wild-Type Virulence of Brucella abortus

Journal of Bacteriology ◽

10.1128/jb.02543-14 ◽

2015 ◽

Vol 197 (9) ◽

pp. 1582-1591 ◽

Cited By ~ 15

Author(s):

Lauren M. Sheehan ◽

James A. Budnick ◽

R. Martin Roop ◽

Clayton C. Caswell

Keyword(s):

Mouse Model ◽

Brucella Abortus ◽

Zinc Uptake ◽

Zinc Homeostasis ◽

Zinc Toxicity ◽

Uptake System ◽

Bacterial Cells ◽

Gene Encoding ◽

Content Type ◽

Genes Encoding

ABSTRACTMetal homeostasis in bacterial cells is a highly regulated process requiring intricately coordinated import and export, as well as precise sensing of intracellular metal concentrations. The uptake of zinc (Zn) has been linked to the virulence ofBrucella abortus; however, the capacity ofBrucellastrains to sense Zn levels and subsequently coordinate Zn homeostasis has not been described. Here, we show that expression of the genes encoding the zinc uptake system ZnuABC is negatively regulated by the Zn-sensing Fur family transcriptional regulator, Zur, by direct interactions between Zur and the promoter region ofznuABC. Moreover, the MerR-type regulator, ZntR, controls the expression of the gene encoding the Zn exporter ZntA by binding directly to its promoter. Deletion ofzurorzntRalone did not result in increased zinc toxicity in the corresponding mutants; however, deletion ofzntAled to increased sensitivity to Zn but not to other metals, such as Cu and Ni, suggesting that ZntA is a Zn-specific exporter. Strikingly, deletion ofzntRresulted in significant attenuation ofB. abortusin a mouse model of chronic infection, and subsequent experiments revealed that overexpression ofzntAin thezntRmutant is the molecular basis for its decreased virulence.IMPORTANCEThe importance of zinc uptake forBrucellapathogenesis has been demonstrated previously, but to date, there has been no description of how overall zinc homeostasis is maintained and genetically controlled in the brucellae. The present work defines the predominant zinc export system, as well as the key genetic regulators of both zinc uptake and export inBrucella abortus. Moreover, the data show the importance of precise coordination of the zinc homeostasis systems as disregulation of some elements of these systems leads to the attenuation ofBrucellavirulence in a mouse model. Overall, this study advances our understanding of the essential role of zinc in the pathogenesis of intracellular bacteria.

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Identification and Characterization of Genes Encoding a Putative ABC-Type Transporter Essential for Utilization of γ-Hexachlorocyclohexane in Sphingobium japonicum UT26

Journal of Bacteriology ◽

10.1128/jb.01883-06 ◽

2007 ◽

Vol 189 (10) ◽

pp. 3712-3720 ◽

Cited By ~ 37

Author(s):

Ryo Endo ◽

Yoshiyuki Ohtsubo ◽

Masataka Tsuda ◽

Yuji Nagata

Keyword(s):

Abc Transporter ◽

Sole Source ◽

Bacterial Cells ◽

New Genes ◽

Hydrophobic Compounds ◽

Dead End ◽

Genes Encoding ◽

Degradation Activity ◽

Mutant Cells

ABSTRACT Sphingobium japonicum UT26 utilizes γ-hexachlorocyclohexane (γ-HCH) as its sole source of carbon and energy. In our previous studies, we cloned and characterized genes encoding enzymes for the conversion of γ-HCH to β-ketoadipate in UT26. In this study, we analyzed a mutant obtained by transposon mutagenesis and identified and characterized new genes encoding a putative ABC-type transporter essential for the utilization of γ-HCH in strain UT26. This putative ABC transporter consists of four components, permease, ATPase, periplasmic protein, and lipoprotein, encoded by linK, linL, linM, and linN, respectively. Mutation and complementation analyses indicated that all the linKLMN genes are required, probably as a set, for γ-HCH utilization in UT26. Furthermore, the mutant cells deficient in this putative ABC transporter showed (i) higher γ-HCH degradation activity and greater accumulation of the toxic dead-end product 2,5-dichlorophenol (2,5-DCP), (ii) higher sensitivity to 2,5-DCP itself, and (iii) higher permeability of hydrophobic compounds than the wild-type cells. These results strongly suggested that LinKLMN are involved in γ-HCH utilization by controlling membrane hydrophobicity. This study clearly demonstrated that a cellular factor besides catabolic enzymes and transcriptional regulators is essential for utilization of xenobiotic compounds in bacterial cells.

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Economy of Operon Formation: Cotranscription Minimizes Shortfall in Protein Complexes

mBio ◽

10.1128/mbio.00177-10 ◽

2010 ◽

Vol 1 (4) ◽

Cited By ~ 17

Author(s):

Kim Sneppen ◽

Steen Pedersen ◽

Sandeep Krishna ◽

Ian Dodd ◽

Szabolcs Semsey

Keyword(s):

Protein Complexes ◽

Small Cells ◽

Eukaryotic Cells ◽

Bacterial Cells ◽

Economic Gain ◽

Eukaryotic Genes ◽

Genes Encoding ◽

Encoding Protein

ABSTRACT Genes of prokaryotes and Archaea are often organized in cotranscribed groups, or operons. In contrast, eukaryotic genes are generally transcribed independently. Here we show that there is a substantial economic gain for the cell to cotranscribe genes encoding protein complexes because it synchronizes the fluctuations, or noise, in the levels of the different components. This correlation substantially reduces the shortfall in production of the complex. This benefit is relatively large in small cells such as bacterial cells, in which there are few mRNAs and proteins per cell, and is diminished in larger cells such as eukaryotic cells.

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Whole-Genome Sequence of Stenotrophomonas maltophilia ZBG7B Reveals Its Biotechnological Potential

Genome Announcements ◽

10.1128/genomea.01442-15 ◽

2015 ◽

Vol 3 (6) ◽

Cited By ~ 3

Author(s):

Kok-Gan Chan ◽

Teik-Min Chong ◽

Tan-Guan-Sheng Adrian ◽

Heng Leong Kher ◽

Kar-Wai Hong ◽

...

Keyword(s):

Genome Sequence ◽

Bacterial Strain ◽

Stenotrophomonas Maltophilia ◽

Draft Genome ◽

Whole Genome Sequence ◽

Whole Genome ◽

Biotechnological Potential ◽

Vineyard Soil ◽

Genes Encoding ◽

Prediction Of Function

Stenotrophomonas maltophilia ZBG7B was isolated from vineyard soil of Zellenberg, France. Here, we present the draft genome sequence of this bacterial strain, which has facilitated the prediction of function for several genes encoding biotechnologically important enzymes, such as xylosidase, xylanase, laccase, and chitinase.

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