Common dyes used to determine bacterial polysaccharides on agar are affected by medium acidification

In this work, we highlight effects of pH on bacterial phenotypes when using the bacteriological dyes Aniline blue, Congo red, and Calcofluor white to analyze polysaccharide production. A study of galactose catabolism in Sinorhizobium meliloti led to the isolation of a mutation in dgoK1, which was observed to overproduce exopolysaccharides when grown in the presence of galactose. When this mutant strain was spotted onto plates containing Aniline blue, Congo red, or Calcofluor white, the intensity of the associated staining was strikingly different from that of the wild type. Additionally, a Calcofluor dull phenotype was observed, suggesting production of a polysaccharide other than succinoglycan. Further investigation of this phenotype revealed that these results were dependent on medium acidification, as buffering at pH 6 had no effect on these phenotypes, while medium buffered at pH 6.5 resulted in a reversal of the phenotypes. Screening for mutants of the dgoK1 strain that were negative for the Aniline blue phenotype yielded a strain carrying a mutation in tkt2, which is annotated as a putative transketolase. Consistent with the plate phenotypes, when this mutant was grown in broth cultures, it did not acidify its growth medium. Overall, this work shows that caution should be exercised in evaluating polysaccharide phenotypes based strictly on the use of dyes.

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The Sinorhizobium meliloti Lon Protease Is Involved in Regulating Exopolysaccharide Synthesis and Is Required for Nodulation of Alfalfa

Journal of Bacteriology ◽

10.1128/jb.182.9.2551-2558.2000 ◽

2000 ◽

Vol 182 (9) ◽

pp. 2551-2558 ◽

Cited By ~ 15

Author(s):

Michael L. Summers ◽

Lina M. Botero ◽

Scott C. Busse ◽

Timothy R. McDermott

Keyword(s):

Mutant Strain ◽

Type Strain ◽

Sinorhizobium Meliloti ◽

Wild Type Strain ◽

Insertion Site ◽

Normal Growth ◽

Phosphate Limitation ◽

Nuclear Magnetic Resonance Analysis ◽

Lon Protease ◽

Wild Type

ABSTRACT While screening for Sinorhizobium meliloti Pho regulatory mutants, a transposon mutant was isolated that constitutively expressed higher levels of acid and alkaline phosphatase enzymes. This mutant was also found to form pseudonodules on alfalfa that were delayed in appearance relative to those formed by the wild-type strain, it contained few bacteroids, and it did not fix nitrogen. Sequence analysis of the transposon insertion site revealed the affected gene to have high homology to Lon proteases from a number of organisms. In minimal succinate medium, the mutant strain was found to grow more slowly, reach lower maximal optical density, and produce more extracellular polysaccharide (EPS) than the wild-type strain. The mutant fluoresced brightly on minimal succinate agar containing calcofluor (which binds to EPSI, a constitutively expressed succinoglycan), and gas chromotographic analysis of purified total EPS showed that the glucose-to-galactose ratio in the lonmutant total EPS was 5.0 ± 0.2 (mean ± standard error), whereas the glucose-to-galactose ratio in the wild-type strain was 7.1 ± 0.5. These data suggested that in addition to EPSI, thelon mutant also constitutively synthesized EPSII, a galactoglucan which is the second major EPS known to be produced byS. meliloti, but typically is expressed only under conditions of phosphate limitation. 13C nuclear magnetic resonance analysis showed no major differences between EPS purified from the mutant and wild-type strains. Normal growth, EPS production, and the symbiotic phenotype were restored in the mutant strain when the wild-type lon gene was present intrans. The results of this study suggest that the S. meliloti Lon protease is important for controlling turnover of a constitutively expressed protein(s) that, when unregulated, disrupts normal nodule formation and normal growth.

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Isolation of a mutant strain of Pseudomonas sp. ATCC 31461 exhibiting elevated polysaccharide production

Journal of Industrial Microbiology & Biotechnology ◽

10.1038/sj/jim/7000278 ◽

2002 ◽

Vol 29 (4) ◽

pp. 185-188

Author(s):

T P West

Keyword(s):

Mutant Strain ◽

Pseudomonas Sp ◽

Polysaccharide Production

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The Lipid Lysyl-Phosphatidylglycerol Is Present in Membranes of Rhizobium tropici CIAT899 and Confers Increased Resistance to Polymyxin B Under Acidic Growth Conditions

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-20-11-1421 ◽

2007 ◽

Vol 20 (11) ◽

pp. 1421-1430 ◽

Cited By ~ 65

Author(s):

Christian Sohlenkamp ◽

Kanaan A. Galindo-Lagunas ◽

Ziqiang Guan ◽

Pablo Vinuesa ◽

Sally Robinson ◽

...

Keyword(s):

Sinorhizobium Meliloti ◽

Minimal Medium ◽

Polymyxin B ◽

Growth Conditions ◽

Membrane Lipid ◽

Low Ph ◽

Wild Type ◽

Rhizobium Tropici ◽

Gram Negative ◽

Inducible Genes

Lysyl-phosphatidylglycerol (LPG) is a well-known membrane lipid in several gram-positive bacteria but is almost unheard of in gram-negative bacteria. In Staphylococcus aureus, the gene product of mprF is responsible for LPG formation. Low pH-inducible genes, termed lpiA, have been identified in the gram-negative α-proteobacteria Rhizobium tropici and Sinorhizobium medicae in screens for acid-sensitive mutants and they encode homologs of MprF. An analysis of the sequenced bacterial genomes reveals that genes coding for homologs of MprF from S. aureus are present in several classes of organisms throughout the bacterial kingdom. In this study, we show that the expression of lpiA from R. tropici in the heterologous hosts Escherichia coli and Sinorhizobium meliloti causes formation of LPG. A wild-type strain of R. tropici forms LPG (about 1% of the total lipids) when the cells are grown in minimal medium at pH 4.5 but not when grown in minimal medium at neutral pH or in complex tryptone yeast (TY) medium at either pH. LPG biosynthesis does not occur when lpiA is deleted and is restored upon complementation of lpiA-deficient mutants with a functional copy of the lpiA gene. When grown in the low-pH medium, lpiA-deficient rhizobial mutants are over four times more susceptible to the cationic peptide polymyxin B than the wild type.

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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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Toxicity of Cadmium and Copper in Chlamydomonas reinhardtii Wild-Type (WT 2137) and Cell Wall Deficient Mutant Strain (CW 15)

Bulletin of Environmental Contamination and Toxicology ◽

10.1007/s001289900626 ◽

1998 ◽

Vol 60 (2) ◽

pp. 306-311 ◽

Cited By ~ 38

Author(s):

M. N. V. Prasad ◽

K. Drej ◽

A. Skawi &#96 ska ◽

K. Stra &#94 ka

Keyword(s):

Cell Wall ◽

Chlamydomonas Reinhardtii ◽

Mutant Strain ◽

Deficient Mutant ◽

Wild Type

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RpoS-Dependent Stress Response and Exoenzyme Production in Vibrio vulnificus

Applied and Environmental Microbiology ◽

10.1128/aem.69.10.6114-6120.2003 ◽

2003 ◽

Vol 69 (10) ◽

pp. 6114-6120 ◽

Cited By ~ 54

Author(s):

A. Hülsmann ◽

T. M. Rosche ◽

I.-S. Kong ◽

H. M. Hassan ◽

D. M. Beam ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Stress Response ◽

Mutant Strain ◽

Environmental Changes ◽

Vibrio Vulnificus ◽

High Sensitivity ◽

Striking Difference ◽

Wild Type ◽

Hydrolytic Activities ◽

In The Wild

ABSTRACT Vibrio vulnificus is an estuarine bacterium capable of causing rapidly fatal infections through both ingestion and wound infection. Like other opportunistic pathogens, V. vulnificus must adapt to potentially stressful environmental changes while living freely in seawater, upon colonization of the oyster gut, and upon infection of such diverse hosts as humans and eels. In order to begin to understand the ability of V. vulnificus to respond to such stresses, we examined the role of the alternate sigma factor RpoS, which is important in stress response and virulence in many pathogens. An rpoS mutant of V. vulnificus strain C7184o was constructed by homologous recombination. The mutant strain exhibited a decreased ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and acidic conditions. The most striking difference was a high sensitivity of the mutant to hydrogen peroxide. Albuminase, caseinase, and elastase activity were detected in the wild type but not in the mutant strain, and an additional two hydrolytic activities (collagenase and gelatinase) were reduced in the mutant strain compared to the wild type. Additionally, the motility of the rpoS mutant was severely diminished. Overall, these studies suggest that rpoS in V. vulnificus is important for adaptation to environmental changes and may have a role in virulence.

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Establishing Virulence Associated Polyphosphate Kinase 2 as a drug target for Mycobacterium tuberculosis

Scientific Reports ◽

10.1038/srep26900 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 13

Author(s):

Mamta Singh ◽

Prabhakar Tiwari ◽

Garima Arora ◽

Sakshi Agarwal ◽

Saqib Kidwai ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Mutant Strain ◽

Guinea Pigs ◽

High Throughput Screening ◽

Drug Target ◽

Wild Type Strain ◽

Wild Type ◽

Inorganic Polyphosphate ◽

Phosphate Donor ◽

Microbial Stress

Abstract Inorganic polyphosphate (PolyP) plays an essential role in microbial stress adaptation, virulence and drug tolerance. The genome of Mycobacterium tuberculosis encodes for two polyphosphate kinases (PPK-1, Rv2984 and PPK-2, Rv3232c) and polyphosphatases (ppx-1, Rv0496 and ppx-2, Rv1026) for maintenance of intracellular PolyP levels. Microbial polyphosphate kinases constitute a molecular mechanism, whereby microorganisms utilize PolyP as phosphate donor for synthesis of ATP. In the present study we have constructed ppk-2 mutant strain of M. tuberculosis and demonstrate that PPK-2 enzyme contributes to its ability to cause disease in guinea pigs. We observed that ppk-2 mutant strain infected guinea pigs had significantly reduced bacterial loads and tissue pathology in comparison to wild type infected guinea pigs at later stages of infection. We also report that in comparison to the wild type strain, ppk-2 mutant strain was more tolerant to isoniazid and impaired for survival in THP-1 macrophages. In the present study we have standardized a luciferase based assay system to identify chemical scaffolds that are non-cytotoxic and inhibit M. tuberculosis PPK-2 enzyme. To the best of our knowledge this is the first study demonstrating feasibility of high throughput screening to obtain small molecule PPK-2 inhibitors.

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RHYTHMS IN BLUE-LIGHT-INDUCED CONIDIATION OF WILD TYPE AND A MUTANT STRAIN OF Trichoderma harzianum

Photochemistry and Photobiology ◽

10.1111/j.1751-1097.1988.tb02747.x ◽

1988 ◽

Vol 47 (3) ◽

pp. 425-431 ◽

Cited By ~ 8

Author(s):

GERALD F. DEITZER ◽

BENJAMIN A. Horwitz ◽

Jonathan Gressel

Keyword(s):

Blue Light ◽

Mutant Strain ◽

Trichoderma Harzianum ◽

Wild Type

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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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