L-Alanine Exporter, AlaE, of Escherichia coli Functions as a Safety Valve to Enhance Survival under Feast Conditions

The intracellular level of amino acids is determined by the balance between their anabolic and catabolic pathways. L-alanine is anabolized by three L-alanine synthesizing enzymes and catabolized by two racemases and D-amino acid dehydrogenase (DadA). In addition, its level is regulated by L-alanine movement across the inner membrane. We identified the novel gene alaE, encoding an L-alanine exporter. To elucidate the physiological function of L-Alanine exporter, AlaE, we determined the susceptibility of alaE-, dadA-, and alaE/dadA-deficient mutants, derived from the wild-type strain MG1655, to L-alanyl-L-alanine (Ala-Ala), which shows toxicity to the L-alanine-nonmetabolizing variant lacking alaE. The dadA-deficient mutant has a similar minimum inhibitory concentration (MIC) (>1.25 mg/mL) to that observed in MG1655. However, alaE- and alaE/dadA-deficient mutants had MICs of 0.04 and 0.0025 mg/mL, respectively. The results suggested that the efficacy of AlaE to relieve stress caused by toxic intracellular accumulation of L-alanine was higher than that of DadA. Consistent with this, the intracellular level of alanine in the alaE-mutant was much higher than that in MG1655 and the dadA-mutant. We, therefore, conclude that AlaE functions as a ‘safety-valve’ to prevent the toxic level accumulation of intracellular L-alanine under a peptide-rich environment, such as within the animal intestine.

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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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Dual Targeting of DNA Gyrase and Topoisomerase IV: Target Interactions of Garenoxacin (BMS-284756, T-3811ME), a New Desfluoroquinolone

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.11.3370-3380.2002 ◽

2002 ◽

Vol 46 (11) ◽

pp. 3370-3380 ◽

Cited By ~ 56

Author(s):

Dilek Ince ◽

Xiamei Zhang ◽

L. Christine Silver ◽

David C. Hooper

Keyword(s):

Inhibitory Concentration ◽

Efflux Pump ◽

Fold Increase ◽

Single Step ◽

The Novel ◽

Wild Type ◽

Topoisomerase Iv ◽

Dual Targeting ◽

Resistant Mutants ◽

Selection Of

ABSTRACT We determined the target enzyme interactions of garenoxacin (BMS-284756, T-3811ME), a novel desfluoroquinolone, in Staphylococcus aureus by genetic and biochemical studies. We found garenoxacin to be four- to eightfold more active than ciprofloxacin against wild-type S. aureus. A single topoisomerase IV or gyrase mutation caused only a 2- to 4-fold increase in the MIC of garenoxacin, whereas a combination of mutations in both loci caused a substantial increase (128-fold). Overexpression of the NorA efflux pump had minimal effect on resistance to garenoxacin. With garenoxacin at twice the MIC, selection of resistant mutants (<7.4 × 10−12 to 4.0 × 10−11) was 5 to 6 log units less than that with ciprofloxacin. Mutations inside or outside the quinolone resistance-determining regions (QRDR) of either topoisomerase IV, or gyrase, or both were selected in single-step mutants, suggesting dual targeting of topoisomerase IV and gyrase. Three of the novel mutations were shown by genetic experiments to be responsible for resistance. Studies with purified topoisomerase IV and gyrase from S. aureus also showed that garenoxacin had similar activity against topoisomerase IV and gyrase (50% inhibitory concentration, 1.25 to 2.5 and 1.25 μg/ml, respectively), and although its activity against topoisomerase IV was 2-fold greater than that of ciprofloxacin, its activity against gyrase was 10-fold greater. This study provides the first genetic and biochemical data supporting the dual targeting of topoisomerase IV and gyrase in S. aureus by a quinolone as well as providing genetic proof for the expansion of the QRDRs to include the 5′ terminus of grlB and the 3′ terminus of gyrA.

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The Transcriptional Antiterminator RfaH Represses Biofilm Formation in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.188.4.1316-1331.2006 ◽

2006 ◽

Vol 188 (4) ◽

pp. 1316-1331 ◽

Cited By ~ 67

Author(s):

Christophe Beloin ◽

Kai Michaelis ◽

Karin Lindner ◽

Paolo Landini ◽

Jörg Hacker ◽

...

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Wild Type Strain ◽

Wild Type ◽

Strain Mg1655 ◽

Upec Strain ◽

E Coli ◽

Initial Adhesion ◽

State Production ◽

K 12

ABSTRACT We investigated the influence of regulatory and pathogenicity island-associated factors (Hha, RpoS, LuxS, EvgA, RfaH, and tRNA5 Leu) on biofilm formation by uropathogenic Escherichia coli (UPEC) strain 536. Only inactivation of rfaH, which encodes a transcriptional antiterminator, resulted in increased initial adhesion and biofilm formation by E. coli 536. rfaH inactivation in nonpathogenic E. coli K-12 isolate MG1655 resulted in the same phenotype. Transcriptome analysis of wild-type strain 536 and an rfaH mutant of this strain revealed that deletion of rfaH correlated with increased expression of flu orthologs. flu encodes antigen 43 (Ag43), which mediates autoaggregation and biofilm formation. We confirmed that deletion of rfaH leads to increased levels of flu and flu-like transcripts in E. coli K-12 and UPEC. Supporting the hypothesis that RfaH represses biofilm formation through reduction of the Ag43 level, the increased-biofilm phenotype of E. coli MG1655rfaH was reversed upon inactivation of flu. Deletion of the two flu orthologs, however, did not modify the behavior of mutant 536rfaH. Our results demonstrate that the strong initial adhesion and biofilm formation capacities of strain MG1655rfaH are mediated by both increased steady-state production of Ag43 and likely increased Ag43 presentation due to null rfaH-dependent lipopolysaccharide depletion. Although the roles of rfaH in the biofilm phenotype are different in UPEC strain 536 and K-12 strain MG1655, this study shows that RfaH, in addition to affecting the expression of bacterial virulence factors, also negatively controls expression and surface presentation of Ag43 and possibly another Ag43-independent factor(s) that mediates cell-cell interactions and biofilm formation.

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Cysteine desulphurase-encoding gene sufS2 is required for the repressor function of RirA and oxidative resistance in Agrobacterium tumefaciens

Microbiology ◽

10.1099/mic.0.068643-0 ◽

2014 ◽

Vol 160 (1) ◽

pp. 79-90 ◽

Cited By ~ 20

Author(s):

Sakkarin Bhubhanil ◽

Phettree Niamyim ◽

Rojana Sukchawalit ◽

Skorn Mongkolsuk

Keyword(s):

Agrobacterium Tumefaciens ◽

Wild Type Strain ◽

Physiological Function ◽

Response Regulator ◽

Cluster Assembly ◽

Wild Type ◽

Oxidative Resistance ◽

Small Colony ◽

Encoding Gene ◽

Assembly Proteins

The Agrobacterium tumefaciens genome contains a cluster of genes that are predicted to encode Fe–S cluster assembly proteins, and this cluster is known as the sufS2BCDS1XA operon. sufS2 is the first gene in the operon, and it was inactivated to determine its physiological function. The sufS2 mutant exhibited a small colony phenotype, grew slower than the wild-type strain and was more sensitive to various oxidants including peroxide, organic hydroperoxide and superoxide. The sufS2 gene was negatively regulated by iron response regulator (Irr) and rhizobial iron regulator (RirA) under low and high iron conditions, respectively, and was inducible in response to oxidative stress. The oxidant-induced expression of sufS2 was controlled by Irr, RirA and an additional but not yet identified mechanism. sufS2 was required for RirA activity in the repression of a sufS2 promoter-lacZ fusion. RirA may use Fe–S as its cofactor. sufS2 disruption may cause a defect in the Fe–S supply and could thereby affect the RirA activity. The three conserved cysteine residues (C91, C99 and C105) in RirA were predicted to coordinate with the Fe–S cluster and were shown to be essential for RirA repression of the sufS2-lacZ fusion. These results suggested that sufS2 is important for the survival of A. tumefaciens.

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Determining the Optimal Thymidine Concentration for Growing Thy−Escherichia coli Strains

Journal of Bacteriology ◽

10.1128/jb.180.11.2992-2994.1998 ◽

1998 ◽

Vol 180 (11) ◽

pp. 2992-2994 ◽

Cited By ~ 10

Author(s):

Felipe Molina ◽

Alfonso Jiménez-Sánchez ◽

Elena C. Guzmán

Keyword(s):

Escherichia Coli ◽

Growth Rate ◽

Growth Medium ◽

Type Strain ◽

Wild Type Strain ◽

Wild Type ◽

Strain Mg1655 ◽

E Coli ◽

Replication Time ◽

Detectable Difference

ABSTRACT Changes of thymidine concentration in the growth medium affect the chromosome replication time of Thy− strains without at the same time causing a detectable difference in the growth rate (R. H. Pritchard and A. Zaritsky, Nature 226:126–131, 1970). Consequently, the optimal thymidine concentration cannot be determined by ascertaining which concentration produces the highest growth rate. Here we present a method for determining the optimal thymidine concentration of any Thy− Escherichia coli strain. Using this method, we found that the E. coli “wild-type” strain MG1655 has a partial Thy− phenotype.

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Characterization of a lipopeptide-resistant strain ofCandida albicans

Canadian Journal of Microbiology ◽

10.1139/m97-016 ◽

1997 ◽

Vol 43 (2) ◽

pp. 122-128 ◽

Cited By ~ 8

Author(s):

David J. Frost ◽

Melinda Knapp ◽

Kim Brandt ◽

Amber Shadron ◽

Robert C. Goldman

Keyword(s):

Candida Albicans ◽

Type Strain ◽

Antifungal Agents ◽

Wild Type Strain ◽

Inhibitory Concentration ◽

Resistance Factor ◽

Wild Type ◽

Glucan Synthase ◽

Resistance Specificity

Lipopeptides are antifungal agents that inhibit cell wall β-(1, 3)-glucan biosynthesis in fungal organisms. A mutant resistant to lipopeptides was generated by UV mutagenesis and characterized. The Candida albicans mutant (LP3-1) was stable and showed resistance specificity to a broad range of lipopeptides and certain glycolipid inhibitors. Other antifungal agents with diverse modes of action had a normal minimum inhibitory concentration profile for LP3-1 compared with the wild-type strain (CCH 442). In the in vitro β-(1, 3)-glucan synthase assay, both the lipopeptides and papulacandin-related agents had considerably higher 50% inhibitory concentration values in the LP3-1 strain than in the wild-type strain. In reconstitution assays, the resistance factor was associated with the integral membrane pellet rather than the peripheral GTP-binding protein. The LP3-1 strain had a membrane lipid profile similar to that of the parent strain and was virulent in a murine model of systemic candidiasis. Taken together, these results indicate that the resistance factor is associated with the integral membrane component of β-(1, 3)-glucan synthase. Lipopeptides are common antifungal agents encountered during screening of natural products. The LP3-1 strain was resistant to natural product extracts known to contain various lipopeptides. Thus, LP3-1 can be used in a dereplication assay.Key words: Candida albicans, β-(1, 3)-glucan synthase, lipopeptides, drug resistance.

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Inducible l-Alanine Exporter Encoded by the Novel GeneygaW(alaE) in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.00003-11 ◽

2011 ◽

Vol 77 (12) ◽

pp. 4027-4034 ◽

Cited By ~ 24

Author(s):

Hatsuhiro Hori ◽

Hiroshi Yoneyama ◽

Ryuta Tobe ◽

Tasuke Ando ◽

Emiko Isogai ◽

...

Keyword(s):

Escherichia Coli ◽

Wild Type Strain ◽

Chemical Mutagenesis ◽

The Novel ◽

Wild Type ◽

Content Type ◽

E Coli ◽

Export Rate ◽

In The Wild ◽

Export System

ABSTRACTWe previously isolated a mutant hypersensitive tol-alanyl-l-alanine from a non-l-alanine-metabolizingEscherichia colistrain and found that it lacked an induciblel-alanine export system. Consequently, this mutant showed a significant accumulation of intracellularl-alanine and a reduction in thel-alanine export rate compared to the parent strain. When the mutant was used as a host to clone a gene(s) that complements the dipeptide-hypersensitive phenotype, two uncharacterized genes,ygaWandytfF, and two characterized genes,yddGandyeaS, were identified. Overexpression of each gene in the mutant resulted in a decrease in the intracellularl-alanine level and enhancement of thel-alanine export rate in the presence of the dipeptide, suggesting that their products function as exporters ofl-alanine. SinceygaWexhibited the most striking impact on both the intra- and the extracellularl-alanine levels among the four genes identified, we disrupted theygaWgene in the non-l-alanine-metabolizing strain. The resulting isogenic mutant showed the same intra- and extracellularl-alanine levels as observed in the dipeptide-hypersensitive mutant obtained by chemical mutagenesis. When each gene was overexpressed in the wild-type strain, which does not intrinsically excrete alanine, only theygaWgene conferred on the cells the ability to excrete alanine. In addition, expression of theygaWgene was induced in the presence of the dipeptide. On the basis of these results, we concluded that YgaW is likely to be the physiologically most relevant exporter forl-alanine inE. coliand proposed that the gene be redesignatedalaEforalanineexport.

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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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Evidence for Establishing the Clinical Breakpoint of Cefquinome against Haemophilus Parasuis in China

Pathogens ◽

10.3390/pathogens10020105 ◽

2021 ◽

Vol 10 (2) ◽

pp. 105

Author(s):

Kun Mi ◽

Da Sun ◽

Mei Li ◽

Haihong Hao ◽

Kaixiang Zhou ◽

...

Keyword(s):

Monte Carlo Simulation ◽

Antibacterial Activity ◽

Inhibitory Concentration ◽

High Morbidity ◽

Haemophilus Parasuis ◽

Wild Type ◽

Time Curve ◽

Resistance Change ◽

Concentration Time ◽

Clinical Experiments

Haemophilus parasuis can cause high morbidity and mortality in swine. Cefquinome possesses excellent antibacterial activity against pathogens causing diseases of the respiratory tract. This study aimed to establish the clinical breakpoint (CBP) of cefquinome against H. parasuis and to monitor the resistance change. Referring to the minimum inhibitory concentration (MIC) distribution of cefquinome against 131 H. parasuis isolates, the MIC50 and MIC90 were determined to be 0.125 and 1 μg/mL, respectively. And the epidemiological cutoff (ECOFF) value was 1 μg/mL. HPS42 was selected as a representative strain for the pharmacodynamic (PD) experiment, pharmacokinetic (PK) experiment and clinical experiments. The PK/PD index values, area under concentration-time curve (AUC)/MIC, of the bacteriostatic, bactericidal, and bacterial elimination effects were 23, 41, and 51 h, respectively. The PK/PD cutoff was calculated as 0.125 μg/mL by Monte Carlo simulation (MCS), and the clinical cutoff was 0.25−4 μg/mL by WindoW. Combing these three values, the CBP of cefquinome against H. parasuis was found to be 1 μg/mL. In conclusion, this was the first study to integrate various cutoffs to establish the CBP in the laboratory. It is helpful to distinguish wild type H. parasuis and reduce the probability of treatment failure.

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