Screen for Leukotoxin Mutants in Aggregatibacter actinomycetemcomitans: Genes of the Phosphotransferase System Are Required for Leukotoxin Biosynthesis

ABSTRACT Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans is a pathogen that causes localized aggressive periodontitis and extraoral infections including infective endocarditis. Recently, we reported that A. actinomycetemcomitans is beta-hemolytic on certain growth media due to the production of leukotoxin (LtxA). Based on this observation and our ability to generate random transposon insertions in A. actinomycetemcomitans, we developed and carried out a rapid screen for LtxA mutants. Using PCR, we mapped several of the mutations to genes that are known or predicted to be required for LtxA production, including ltxA, ltxB, ltxD, and tdeA. In addition, we identified an insertion in a gene previously not recognized to be involved in LtxA biosynthesis, ptsH. ptsH encodes the protein HPr, a phosphocarrier protein that is part of the sugar phosphotransferase system. HPr results in the phosphorylation of other proteins and ultimately in the activation of adenylate cyclase and cyclic AMP (cAMP) production. The ptsH mutant showed only partial hemolysis on blood agar and did not produce LtxA. The phenotype was complemented by supplying wild-type ptsH in trans, and real-time PCR analysis showed that the ptsH mutant produced approximately 10-fold less ltxA mRNA than the wild-type strain. The levels of cAMP in the ptsH mutant were significantly lower than in the wild-type strain, and LtxA production could be restored by adding exogenous cAMP to the culture.

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Characterization of γ-Butyrolactone Autoregulatory Signaling Gene Homologs in the Angucyclinone Polyketide WS5995B Producer Streptomyces acidiscabies

Journal of Bacteriology ◽

10.1128/jb.00437-09 ◽

2009 ◽

Vol 191 (15) ◽

pp. 4786-4797 ◽

Cited By ~ 24

Author(s):

Frank G. Healy ◽

Kevin P. Eaton ◽

Prajit Limsirichai ◽

Joel F. Aldrich ◽

Alaina K. Plowman ◽

...

Keyword(s):

Dna Sequences ◽

Type Strain ◽

Wild Type Strain ◽

Morphological Differentiation ◽

Pcr Analysis ◽

Wild Type ◽

Reverse Transcription Pcr ◽

Morphological Defects ◽

In The Wild ◽

Streptomyces Acidiscabies

ABSTRACT Organisms belonging to the genus Streptomyces produce numerous important secondary metabolites and undergo a sophisticated morphological differentiation program. In many instances these processes are under the control of γ-butyrolactone (GBL) autoregulatory systems. Streptomyces acidiscabies strain 84.104 produces the secondary metabolite aromatic angucyclinone polyketide WS5995B. In order to explore the role of GBL regulatory circuitry in WS5995B production and morphogenesis in S. acidiscabies, a gene cluster encoding GBL autoregulatory signaling homologs was identified and characterized. Two GBL receptor homologs, sabR and sabS, were found flanking a GBL synthase homolog sabA. Strains carrying mutations in sabS produced elevated levels of WS5995B and displayed conditional morphological defects reminiscent of defects seen in Streptomyces bldA mutants. Notably, sabS possesses a TTA codon predicted to be recognized by tRNAleu. sabA mutants produced higher levels of WS5995B than the wild-type strain but to a lesser extent than the levels of WS5995B seen in sabS mutants. Purified recombinant SabR and SabS were tested for their abilities to bind predicted AT-rich autoregulatory element (ARE) boxes within the sabRAS region. SabS did not bind any DNA sequences in this region, while SabR bound an ARE box in the region upstream of sabS. Quantitative reverse transcription-PCR analysis revealed higher levels of sabS transcript in sabR mutants than in the wild-type strain, suggesting that sabS expression is repressed by SabR. Based on these data, we propose that the S. acidiscabies sabRAS genes encode components of a signaling pathway which participates in the regulation of WS5995B production and morphogenesis.

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Interference of Components of the Phosphoenolpyruvate Phosphotransferase System with the Central Virulence Gene Regulator PrfA of Listeria monocytogenes

Journal of Bacteriology ◽

10.1128/jb.00972-06 ◽

2006 ◽

Vol 189 (2) ◽

pp. 473-490 ◽

Cited By ~ 75

Author(s):

Sonja Mertins ◽

Biju Joseph ◽

Monika Goetz ◽

Regina Ecke ◽

Gerald Seidel ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Type Strain ◽

Virulence Genes ◽

Wild Type Strain ◽

Virulence Gene ◽

Wild Type ◽

Phosphotransferase System ◽

In The Wild ◽

The Common ◽

Transcript Profiles

ABSTRACT Analysis of Listeria monocytogenes ptsH, hprK, and ccpA mutants defective in carbon catabolite repression (CCR) control revealed significant alterations in the expression of PrfA-dependent genes. The hprK mutant showed high up-regulation of PrfA-dependent virulence genes upon growth in glucose-containing medium whereas expression of these genes was even slightly down-regulated in the ccpA mutant compared to the wild-type strain. The ptsH mutant could only grow in a rich culture medium, and here the PrfA-dependent genes were up-regulated as in the hprK mutant. As expected, HPr-Ser-P was not produced in the hprK and ptsH mutants and synthesized at a similar level in the ccpA mutant as in the wild-type strain. However, no direct correlation was found between the level of HPr-Ser-P or HPr-His-P and PrfA activity when L. monocytogenes was grown in minimal medium with different phosphotransferase system (PTS) carbohydrates. Comparison of the transcript profiles of the hprK and ccpA mutants with that of the wild-type strain indicates that the up-regulation of the PrfA-dependent virulence genes in the hprK mutant correlates with the down-regulation of genes known to be controlled by the efficiency of PTS-mediated glucose transport. Furthermore, growth in the presence of the non-PTS substrate glycerol results in high PrfA activity. These data suggest that it is not the component(s) of the CCR or the common PTS pathway but, rather, the component(s) of subsequent steps that seem to be involved in the modulation of PrfA activity.

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Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from Lactobacillus casei Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion

Journal of Bacteriology ◽

10.1128/jb.182.9.2582-2590.2000 ◽

2000 ◽

Vol 182 (9) ◽

pp. 2582-2590 ◽

Cited By ~ 60

Author(s):

Valérie Dossonnet ◽

Vicente Monedero ◽

Monique Zagorec ◽

Anne Galinier ◽

Gaspar Pérez-Martínez ◽

...

Keyword(s):

Catabolite Repression ◽

Type Strain ◽

Lactobacillus Casei ◽

Wild Type Strain ◽

Carbon Sources ◽

Lag Phase ◽

Wild Type ◽

Phosphotransferase System ◽

Inducer Exclusion ◽

In The Wild

ABSTRACT We have cloned and sequenced the Lactobacillus casei hprK gene encoding the bifunctional enzyme HPr kinase/P-Ser-HPr phosphatase (HprK/P). Purified recombinant L. casei HprK/P catalyzes the ATP-dependent phosphorylation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system at the regulatory Ser-46 as well as the dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr). The two opposing activities of HprK/P were regulated by fructose-1,6-bisphosphate, which stimulated HPr phosphorylation, and by inorganic phosphate, which stimulated the P-Ser-HPr phosphatase activity. A mutant producing truncated HprK/P was found to be devoid of both HPr kinase and P-Ser-HPr phosphatase activities. When hprK was inactivated, carbon catabolite repression of N-acetylglucosaminidase disappeared, and the lag phase observed during diauxic growth of the wild-type strain on media containing glucose plus either lactose or maltose was strongly diminished. In addition, inducer exclusion exerted by the presence of glucose on maltose transport in the wild-type strain was abolished in the hprK mutant. However, inducer expulsion ofmethyl β-d-thiogalactoside triggered by rapidly metabolizable carbon sources was still operative inptsH mutants altered at Ser-46 of HPr and thehprK mutant, suggesting that, in contrast to the model proposed for inducer expulsion in gram-positive bacteria, P-Ser-HPr might not be involved in this regulatory process.

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Novel Na+/H+ antiporter (NapA) regulates the motility in Helicobacter pylori

GSC Advanced Research and Reviews ◽

10.30574/gscarr.2021.8.3.0188 ◽

2021 ◽

Vol 8 (3) ◽

pp. 027-035

Author(s):

Masaaki Minami ◽

Shin-nosuke Hashikawa ◽

Takafumi Ando ◽

Hiroshi Kobayashi ◽

Hidemi Goto ◽

...

Keyword(s):

Helicobacter Pylori ◽

Mutant Strain ◽

Type Strain ◽

Wild Type Strain ◽

Pcr Analysis ◽

Wild Type ◽

Cellular Homeostasis ◽

In The Wild ◽

Flagellar Proteins ◽

H Pylori

Na+/H+ antiporter plays an important role in maintaining cellular homeostasis by regulating osmotic pressure and intracellular pH. It plays an important role in maintaining cellular homeostasis. In Helicobacter pylori, whole genome sequencing has revealed the presence of two types of Na+/H+ antiporter. A gene (nhaA) homologous to the Na+/H+ antiporter of Escherichia coli has been investigated and its function has been analyzed. However, another gene homologous to the Na+/H+ antiporter of Enterococcus hirae (napA) is not yet known in detail. In this study, we investigated the function of this gene (napA in H. pylori). First, to confirm the genetic presence of napA in 20 H. pylori clinical isolates, PCR analysis was performed, and the napA gene was confirmed in all strains. The amount of Na+ extrusion was measured by atomic absorption spectroscopy. The results showed that the Na+ concentration was decreased in the wild-type strain compared to the napA mutant strain. In addition, there was a significant dose-dependent difference in CFU of Na+ concentration in the napA mutant strain compared to the wild-type strain. We examined whether the napA gene is related to motility using both wild-type and napA mutant strains. As a result, in the motility agar test, the bacterial motility observed in the wild-type strain was not observed in the napA mutant strain. However, no difference in flagellar proteins was observed by SDS-PAGE analysis. These results suggest that the napA gene of H. pylori may regulate homeostasis by extruding Na+ and may also regulate motility.

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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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Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

Scientific Reports ◽

10.1038/s41598-020-80125-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

José Francisco Cruz-Pérez ◽

Roxana Lara-Oueilhe ◽

Cynthia Marcos-Jiménez ◽

Ricardo Cuatlayotl-Olarte ◽

María Luisa Xiqui-Vázquez ◽

...

Keyword(s):

Azospirillum Brasilense ◽

Type Strain ◽

Wild Type Strain ◽

Soil Conditions ◽

Wild Type ◽

Gene Encoding ◽

Wheat Roots ◽

Genes Encoding ◽

In The Wild ◽

Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

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The phosphoenolpyruvate: sugar phosphotransferase system of Streptococcus salivarius. Identification of a IIIman protein

Canadian Journal of Microbiology ◽

10.1139/m87-020 ◽

1987 ◽

Vol 33 (2) ◽

pp. 118-122 ◽

Cited By ~ 14

Author(s):

Christian Vadeboncoeur ◽

Lucie Gauthier

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Deae Cellulose ◽

Reaction Medium ◽

Inhibitory Effect ◽

Spontaneous Mutant ◽

Streptococcus Salivarius ◽

Wild Type ◽

Phosphotransferase System ◽

Growth Inhibitory

A double-spontaneous mutant resistant to the growth inhibitory effect of α-methylglucoside and 2-deoxyglucose was isolated from Streptococcus salivarius. This mutant strain, called αS3L11, did not grow on mannose and grew poorly on 5 mM fructose and 5 mM glucose. Isolated membranes of strain αS3L11 were unable to catalyse the phosphoenolpyruvate-dependent phosphorylation of mannose in the presence of purified enzyme I and HPr. Addition of dialysed membrane-free cellular extract of the wild-type strain to the reaction medium restored the activity. The factor that restored the phosphoenolpyruvate–mannose phosphotransferase activity to membranes of strain αS3L11 was called IIIman. This factor was partially purified from the wild-type strain by DEAE-cellulose chromatography, DEAE-TSK chromatography, and molecular seiving on a column of Ultrogel AcA 34. This partially purified preparation also enhanced the phosphoenolpyruvate-dependent phosphorylation of glucose, fructose, and 2-deoxyglucose in strain αS3L11.

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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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Disruption of the RAD52 gene alters the spectrum of spontaneous SUP4-o mutations in Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/122.3.535 ◽

1989 ◽

Vol 122 (3) ◽

pp. 535-542 ◽

Cited By ~ 1

Author(s):

B A Kunz ◽

M G Peters ◽

S E Kohalmi ◽

J D Armstrong ◽

M Glattke ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Base Pair ◽

Type Strain ◽

Wild Type Strain ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Mutator Phenotype ◽

In The Wild ◽

Single Base Pair ◽

Almost All

Abstract Defects in the RAD52 gene of the yeast Saccharomyces cerevisiae confer a mutator phenotype. To characterize this effect in detail, a collection of 238 spontaneous SUP4-o mutations arising in a strain having a disrupted RAD52 gene was analyzed by DNA sequencing. The resulting mutational spectrum was compared to that derived from an examination of 222 spontaneous mutations selected in a nearisogenic wild-type (RAD52) strain. This comparison revealed that the mutator phenotype was associated with an increase in the frequency of base-pair substitutions. All possible types of substitution were detected but there was a reduction in the relative fraction of A.T----G.C transitions and an increase in the proportion of G.C----C.G transversions. These changes were sufficient to cause a twofold greater preference for substitutions at G.C sites in the rad52 strain despite a decrease in the fraction of G.C----T.A transversions. There were also considerable differences between the distributions of substitutions within the SUP4-o gene. Base-pair changes occurred at fewer sites in the rad52 strain but the mutated sites included several that were not detected in the RAD52 background. Only two of the four sites that were mutated most frequently in the rad52 strain were also prominent in the wild-type strain and mutation frequencies at almost all sites common to both strains were greater for the rad52 derivative. Although single base-pair deletions occurred in the two strains with similar frequencies, several classes of mutation that were recovered in the wild-type background including multiple base-pair deletions, insertions of the yeast transposable element Ty, and more complex changes, were not detected in the rad52 strain.(ABSTRACT TRUNCATED AT 250 WORDS)

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Plasmid DNA Production in Proteome-Reduced Escherichia coli

Microorganisms ◽

10.3390/microorganisms8091444 ◽

2020 ◽

Vol 8 (9) ◽

pp. 1444

Author(s):

Mitzi de la Cruz ◽

Elisa A. Ramírez ◽

Juan-Carlos Sigala ◽

José Utrilla ◽

Alvaro R. Lara

Keyword(s):

Escherichia Coli ◽

Plasmid Dna ◽

Type Strain ◽

Wild Type Strain ◽

The Novel ◽

Wild Type ◽

Batch Mode ◽

Cell Factories ◽

Starting Point ◽

In The Wild

The design of optimal cell factories requires engineering resource allocation for maximizing product synthesis. A recently developed method to maximize the saving in cell resources released 0.5% of the proteome of Escherichia coli by deleting only three transcription factors. We assessed the capacity for plasmid DNA (pDNA) production in the proteome-reduced strain in a mineral medium, lysogeny, and terrific broths. In all three cases, the pDNA yield from biomass was between 33 and 53% higher in the proteome-reduced than in its wild type strain. When cultured in fed-batch mode in shake-flask, the proteome-reduced strain produced 74.8 mg L−1 pDNA, which was four times greater than its wild-type strain. Nevertheless, the pDNA supercoiled fraction was less than 60% in all cases. Deletion of recA increased the pDNA yields in the wild type, but not in the proteome-reduced strain. Furthermore, recA mutants produced a higher fraction of supercoiled pDNA, compared to their parents. These results show that the novel proteome reduction approach is a promising starting point for the design of improved pDNA production hosts.

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