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

ABSTRACT Sequencing of the genome of Ralstonia solanacearum revealed several genes that putatively code for polyphenol oxidases (PPOs). To study the actual expression of these genes, we looked for and detected all kinds of PPO activities, including laccase, cresolase, and catechol oxidase activities, in cellular extracts of this microorganism. The conditions for the PPO assays were optimized for the phenolic substrate, pH, and sodium dodecyl sulfate concentration used. It was demonstrated that three different PPOs are expressed. The genes coding for the enzymes were unambiguously correlated with the enzymatic activities detected by generation of null mutations in the genes by using insertional mutagenesis with a suicide plasmid and estimating the changes in the levels of enzymatic activities compared to the levels in the wild-type strain. The protein encoded by the RSp1530 locus is a multicopper protein with laccase activity. Two other genes, RSc0337 and RSc1501, code for nonblue copper proteins exhibiting homology to tyrosinases. The product of RSc0337 has strong tyrosine hydroxylase activity, and it has been shown that this enzyme is involved in melanin synthesis by R. solanacearum. The product of the RSc1501 gene is an enzyme that shows a clear preference for oxidation of o-diphenols. Preliminary characterization of the mutants obtained indicated that PPOs expressed by R. solanacearum may participate in resistance to phenolic compounds since the mutants exhibited higher sensitivity to l-tyrosine than the wild-type strain. These results suggest a possible role in the pathogenic process to avoid plant resistance mechanisms involving the participation of phenolic compounds.

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Trehalose Synthesis Contributes to Osmotic Stress Tolerance and Virulence of the Bacterial Wilt Pathogen Ralstonia solanacearum

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-08-19-0218-r ◽

2020 ◽

Vol 33 (3) ◽

pp. 462-473 ◽

Cited By ~ 3

Author(s):

April M. MacIntyre ◽

John X. Barth ◽

Molly C. Pellitteri Hahn ◽

Cameron O. Scarlett ◽

Stéphane Genin ◽

...

Keyword(s):

Osmotic Stress ◽

Bacterial Wilt ◽

Ralstonia Solanacearum ◽

Type Strain ◽

Wild Type Strain ◽

Xylem Sap ◽

Wilt Disease ◽

Wild Type ◽

Tomato Plants ◽

Trehalose Synthesis

The xylem-dwelling plant pathogen Ralstonia solanacearum changes the chemical composition of host xylem sap during bacterial wilt disease. The disaccharide trehalose, implicated in stress tolerance across all kingdoms of life, is enriched in sap from R. solanacearum–infected tomato plants. Trehalose in xylem sap could be synthesized by the bacterium, the plant, or both. To investigate the source and role of trehalose metabolism during wilt disease, we evaluated the effects of deleting the three trehalose synthesis pathways in the pathogen: TreYZ, TreS, and OtsAB, as well as its sole trehalase, TreA. A quadruple treY/treS/otsA/treA mutant produced 30-fold less intracellular trehalose than the wild-type strain missing the trehalase enzyme. This trehalose-nonproducing mutant had reduced tolerance to osmotic stress, which the bacterium likely experiences in plant xylem vessels. Following naturalistic soil-soak inoculation of tomato plants, this triple mutant did not cause disease as well as wild-type R. solanacearum. Further, the wild-type strain out-competed the trehalose-nonproducing mutant by over 600-fold when tomato plants were coinoculated with both strains, showing that trehalose biosynthesis helps R. solanacearum overcome environmental stresses during infection. An otsA (trehalose-6-phosphate synthase) single mutant behaved similarly to ΔtreY/treS/otsA in all experimental settings, suggesting that the OtsAB pathway is the dominant trehalose synthesis pathway in R. solanacearum.

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Relationship of Wild Type Strain Motility and Interaction with Host Plants in Ralstonia solanacearum

Bacterial Wilt Disease ◽

10.1007/978-3-662-03592-4_27 ◽

1998 ◽

pp. 184-191 ◽

Cited By ~ 1

Author(s):

G. Z. Mao ◽

L. Y. He

Keyword(s):

Ralstonia Solanacearum ◽

Type Strain ◽

Host Plants ◽

Wild Type Strain ◽

Wild Type ◽

Relationship Of

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Chemotaxis Is Required for Virulence and Competitive Fitness of the Bacterial Wilt Pathogen Ralstonia solanacearum

Journal of Bacteriology ◽

10.1128/jb.188.10.3697-3708.2006 ◽

2006 ◽

Vol 188 (10) ◽

pp. 3697-3708 ◽

Cited By ~ 177

Author(s):

Jian Yao ◽

Caitilyn Allen

Keyword(s):

Host Plant ◽

Plant Pathogen ◽

Ralstonia Solanacearum ◽

Type Strain ◽

Wild Type Strain ◽

Random Motion ◽

Wild Type ◽

Tomato Plants ◽

Swimming Motility ◽

Directed Motility

ABSTRACT Ralstonia solanacearum, a soilborne plant pathogen of considerable economic importance, invades host plant roots from the soil. Qualitative and quantitative chemotaxis assays revealed that this bacterium is specifically attracted to diverse amino acids and organic acids, and especially to root exudates from the host plant tomato. Exudates from rice, a nonhost plant, were less attractive. Eight different strains from this heterogeneous species complex varied significantly in their attraction to a panel of carbohydrate stimuli, raising the possibility that chemotactic responses may be differentially selected traits that confer adaptation to various hosts or ecological conditions. Previous studies found that an aflagellate mutant lacking swimming motility is significantly reduced in virulence, but the role of directed motility mediated by the chemotaxis system was not known. Two site-directed R. solanacearum mutants lacking either CheA or CheW, which are core chemotaxis signal transduction proteins, were completely nonchemotactic but retained normal swimming motility. In biologically realistic soil soak virulence assays on tomato plants, both nonchemotactic mutants had significantly reduced virulence indistinguishable from that of a nonmotile mutant, demonstrating that directed motility, not simply random motion, is required for full virulence. In contrast, nontactic strains were as virulent as the wild-type strain was when bacteria were introduced directly into the plant stem through a cut petiole, indicating that taxis makes its contribution to virulence in the early stages of host invasion and colonization. When inoculated individually by soaking the soil, both nontactic mutants reached the same population sizes as the wild type did in the stems of tomato plants just beginning to wilt. However, when tomato plants were coinoculated with a 1:1 mixture of a nontactic mutant and its wild-type parent, the wild-type strain outcompeted both nontactic mutants by 100-fold. Together, these results indicate that chemotaxis is an important trait for virulence and pathogenic fitness in this plant pathogen.

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Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/142.2.383 ◽

1996 ◽

Vol 142 (2) ◽

pp. 383-391 ◽

Cited By ~ 2

Author(s):

Yasumasa Tsukamoto ◽

Jun-ichi Kato ◽

Hideo Ikeda

Keyword(s):

Saccharomyces Cerevisiae ◽

Quantitative Analysis ◽

Structural Analysis ◽

Recombination Rate ◽

Type Strain ◽

Negative Selection ◽

Wild Type Strain ◽

Illegitimate Recombination ◽

Wild Type ◽

In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

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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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FixJ family regulator AcfR of Azorhizobium caulinodans is involved in symbiosis with the host plant

BMC Microbiology ◽

10.1186/s12866-021-02138-w ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Wei Liu ◽

Xue Bai ◽

Yan Li ◽

Haikun Zhang ◽

Xiaoke Hu

Keyword(s):

Signal Transduction ◽

Host Plant ◽

Type Strain ◽

Wild Type Strain ◽

Response Regulator ◽

Azorhizobium Caulinodans ◽

Wild Type ◽

Response Regulators ◽

Free Living ◽

Two Component

Abstract Background A wide variety of bacterial adaptative responses to environmental conditions are mediated by signal transduction pathways. Two-component signal transduction systems are one of the predominant means used by bacteria to sense the signals of the host plant and adjust their interaction behaviour. A total of seven open reading frames have been identified as putative two-component response regulators in the gram-negative nitrogen-fixing bacteria Azorhizobium caulinodans ORS571. However, the biological functions of these response regulators in the symbiotic interactions between A. caulinodans ORS571 and the host plant Sesbania rostrata have not been elucidated to date. Results In this study, we identified and investigated a two-component response regulator, AcfR, with a phosphorylatable N-terminal REC (receiver) domain and a C-terminal HTH (helix-turn-helix) LuxR DNA-binding domain in A. caulinodans ORS571. Phylogenetic analysis showed that AcfR possessed close evolutionary relationships with NarL/FixJ family regulators. In addition, six histidine kinases containing HATPase_c and HisKA domains were predicted to interact with AcfR. Furthermore, the biological function of AcfR in free-living and symbiotic conditions was elucidated by comparing the wild-type strain and the ΔacfR mutant strain. In the free-living state, the cell motility behaviour and exopolysaccharide production of the ΔacfR mutant were significantly reduced compared to those of the wild-type strain. In the symbiotic state, the ΔacfR mutant showed a competitive nodule defect on the stems and roots of the host plant, suggesting that AcfR can provide A. caulinodans with an effective competitive ability for symbiotic nodulation. Conclusions Our results showed that AcfR, as a response regulator, regulates numerous phenotypes of A. caulinodans under the free-living conditions and in symbiosis with the host plant. The results of this study help to elucidate the involvement of a REC + HTH_LuxR two-component response regulator in the Rhizobium-host plant interaction.

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Mab_3083c Is a Homologue of RNase J and Plays a Role in Colony Morphotype, Aggregation, and Sliding Motility of Mycobacterium abscessus

Microorganisms ◽

10.3390/microorganisms9040676 ◽

2021 ◽

Vol 9 (4) ◽

pp. 676

Author(s):

Ting-Yu Liu ◽

Sheng-Hui Tsai ◽

Jenn-Wei Chen ◽

Yu-Ching Wang ◽

Shiau-Ting Hu ◽

...

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Opportunistic Pathogen ◽

Mycobacterium Abscessus ◽

Colony Morphology ◽

Clinical Strain ◽

Immunocompromised Patients ◽

Clinical Infection ◽

Wild Type ◽

Sliding Motility

Mycobacterium abscessus is an opportunistic pathogen causing human diseases, especially in immunocompromised patients. M. abscessus strains with a rough morphotype are more virulent than those with a smooth morphotype. Morphotype switch may occur during a clinical infection. To investigate the genes involved in colony morphotype switching, we performed transposon mutagenesis in a rough clinical strain of M. abscessus. A morphotype switching mutant (smooth) named mab_3083c::Tn was obtained. This mutant was found to have a lower aggregative ability and a higher sliding motility than the wild type strain. However, its glycopeptidolipid (GPL) content remained the same as those of the wild type. Complementation of the mutant with a functional mab_3083c gene reverted its morphotype back to rough, indicating that mab_3083c is associated with colony morphology of M. abscessus. Bioinformatic analyses showed that mab_3083c has a 75.4% identity in amino acid sequence with the well-characterized ribonuclease J (RNase J) of M. smegmatis (RNase JMsmeg). Complementation of the mutant with the RNase J gene of M. smegmatis also switched its colony morphology from smooth back to rough. These results suggest that Mab_3083c is a homologue of RNase J and involved in regulating M. abscessus colony morphotype switching.

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Mutations of folC cause increased susceptibility to sulfamethoxazole in Mycobacterium tuberculosis

Scientific Reports ◽

10.1038/s41598-020-80213-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ruiqi Wang ◽

Kun Li ◽

Jifang Yu ◽

Jiaoyu Deng ◽

Yaokai Chen

Keyword(s):

Mycobacterium Tuberculosis ◽

Type Strain ◽

Wild Type Strain ◽

Clinical Isolates ◽

Aminobenzoic Acid ◽

Wild Type ◽

Dihydropteroate Synthase ◽

Expression Levels ◽

Encoding Gene ◽

Increased Susceptibility

AbstractPrevious studies showed that mutation of folC caused decreased expression of the dihydropteroate synthase encoding gene folP2 in Mycobacterium tuberculosis (M. tuberculosis). We speculated that mutation of folC in M. tuberculosis might affect the susceptibility to sulfamethoxazole (SMX). To prove this, 53 clinical isolates with folC mutations were selected and two folC mutants (I43A, I43T) were constructed based on M. tuberculosis H37Ra. The results showed that 42 of the 53 clinical isolates (79.2%) and the two lab-constructed folC mutants were more sensitive to SMX. To probe the mechanism by which folC mutations make M. tuberculosis more sensitive to SMX, folP2 was deleted in H37Ra, and expression levels of folP2 were compared between H37Ra and the two folC mutants. Although deletion of folP2 resulted in increased susceptibility to SMX, no difference in folP2 expression was observed. Furthermore, production levels of para-aminobenzoic acid (pABA) were compared between the folC mutants and the wild-type strain, and results showed that folC mutation resulted in decreased production of pABA. Taken together, we show that folC mutation leads to decreased production of pABA in M. tuberculosis and thus affects its susceptibility to SMX, which broadens our understanding of mechanisms of susceptibilities to antifolates in this bacterium.

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