scholarly journals Role of the ssu and seu Genes of Corynebacterium glutamicum ATCC 13032 in Utilization of Sulfonates and Sulfonate Esters as Sulfur Sources

2005 ◽  
Vol 71 (10) ◽  
pp. 6104-6114 ◽  
Author(s):  
D. J. Koch ◽  
C. Rückert ◽  
D. A. Rey ◽  
A. Mix ◽  
A. Pühler ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Sequence Similarity ◽  
Gene Clusters ◽  
Flavin Mononucleotide ◽  
Gene Products ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Mutant Strains ◽  
Similarity Searches

ABSTRACT Corynebacterium glutamicum ATCC 13032 was found to be able to utilize a broad range of sulfonates and sulfonate esters as sulfur sources. The two gene clusters potentially involved in sulfonate utilization, ssuD1CBA and ssuI-seuABC-ssuD2, were identified in the genome of C. glutamicum ATCC 13032 by similarity searches. While the ssu genes encode proteins resembling Ssu proteins from Escherichia coli or Bacillus subtilis, the seu gene products exhibited similarity to the dibenzothiophene-degrading Dsz monooxygenases of Rhodococcus strain IGTS8. Growth tests with the C. glutamicum wild-type and appropriate mutant strains showed that the clustered genes ssuC, ssuB, and ssuA, putatively encoding the components of an ABC-type transporter system, are required for the utilization of aliphatic sulfonates. In C. glutamicum sulfonates are apparently degraded by sulfonatases encoded by ssuD1 and ssuD2. It was also found that the seu genes seuA, seuB, and seuC can effectively replace ssuD1 and ssuD2 for the degradation of sulfonate esters. The utilization of all sulfonates and sulfonate esters tested is dependent on a novel putative reductase encoded by ssuI. Obviously, all monooxygenases encoded by the ssu and seu genes, including SsuD1, SsuD2, SeuA, SeuB, and SeuC, which are reduced flavin mononucleotide dependent according to sequence similarity, have SsuI as an essential component. Using real-time reverse transcription-PCR, the ssu and seu gene cluster was found to be expressed considerably more strongly during growth on sulfonates and sulfonate esters than during growth on sulfate.

scholarly journals Blocks in the pseudouridimycin pathway unlock hidden metabolites in the Streptomyces producer strain

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marianna Iorio ◽  
Sahar Davatgarbenam ◽  
Stefania Serina ◽  
Paolo Criscenzo ◽  
Mitja M. Zdouc ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Systematic Investigation ◽  
Biosynthetic Gene ◽  
Wild Type ◽  
Bacterial Rna ◽  
Soil Isolate ◽  
Mutant Strains ◽  
In The Wild ◽  
Polymerase Inhibitor

AbstractWe report a metabolomic analysis of Streptomyces sp. ID38640, a soil isolate that produces the bacterial RNA polymerase inhibitor pseudouridimycin. The analysis was performed on the wild type, on three newly constructed and seven previously reported mutant strains disabled in different genes required for pseudouridimycin biosynthesis. The results indicate that Streptomyces sp. ID38640 is able to produce, in addition to lydicamycins and deferroxiamines, as previously reported, also the lassopeptide ulleungdin, the non-ribosomal peptide antipain and the osmoprotectant ectoine. The corresponding biosynthetic gene clusters were readily identified in the strain genome. We also detected the known compound pyridindolol, for which we propose a previously unreported biosynthetic gene cluster, as well as three families of unknown metabolites. Remarkably, the levels of most metabolites varied strongly in the different mutant strains, an observation that enabled detection of metabolites unnoticed in the wild type. Systematic investigation of the accumulated metabolites in the ten different pum mutants identified shed further light on pseudouridimycin biosynthesis. We also show that several Streptomyces strains, able to produce pseudouridimycin, have distinct genetic relationship and metabolic profile with ID38640.

scholarly journals Polysaccharides and virulence of Burkholderia pseudomallei

2007 ◽  
Vol 56 (8) ◽  
pp. 1005-1010 ◽  
Author(s):  
M. Sarkar-Tyson ◽  
J. E. Thwaite ◽  
S. V. Harding ◽  
S. J. Smither ◽  
P. C. F. Oyston ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Capsular Polysaccharide ◽  
Gene Clusters ◽  
Type I ◽  
Wild Type ◽  
Time To Death ◽  
Type Iii ◽  
Type Iv ◽  
Mutant Strains ◽  
Delayed Time

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease of humans and animals. Gene clusters which encode capsular polysaccharide (type I O-PS) and LPS (type II O-PS), both of which play roles in virulence, have previously been identified. Here, the identification of two further putative clusters, type III O-PS and type IV O-PS, is reported. Mice challenged with type III O-PS or type IV O-PS mutants showed increased mean times to death (7.8 and 11.6 days) compared to those challenged with wild-type B. pseudomallei (3 days). To investigate the possible roles of polysaccharides in protection, mice were immunized with killed cells of wild-type B. pseudomallei or killed cells of B. pseudomallei with mutations in the O antigen, capsular polysaccharide, type III O-PS or type IV O-PS gene clusters. Immunization with all polysaccharide mutant strains resulted in delayed time to death compared to the naïve controls, following challenge with wild-type B. pseudomallei strain K96243. However, immunization with killed polysaccharide mutant strains conferred different degrees of protection, demonstrating the immunological importance of the polysaccharide clusters on the surface of B. pseudomallei.

scholarly journals Pneumococcal Surface Protein C Contributes to Sepsis Caused by Streptococcus pneumoniae in Mice

2004 ◽  
Vol 72 (5) ◽  
pp. 3077-3080 ◽  
Author(s):  
Francesco Iannelli ◽  
Damiana Chiavolini ◽  
Susanna Ricci ◽  
Marco Rinaldo Oggioni ◽  
Gianni Pozzi
Keyword(s):  
Streptococcus Pneumoniae ◽  
Protein C ◽  
Lethal Dose ◽  
Surface Protein ◽  
Infection Model ◽  
Wild Type ◽  
Mutant Strains ◽  
Type 3

ABSTRACT The role of pneumococcal surface protein C (PspC; also called SpsA, CbpA, and Hic) in sepsis by Streptococcus pneumoniae was investigated in a murine infection model. The pspC gene was deleted in strains D39 (type 2) and A66 (type 3), and the mutants were tested by being injected intravenously into mice. The animals infected with the mutant strains showed a significant increase in survival, with the 50% lethal dose up to 250-fold higher than that for the wild type. Our findings indicate that PspC affords a decisive contribution to sepsis development.

scholarly journals Expression of Mycobacterium smegmatisPyrazinamidase in Mycobacterium tuberculosis Confers Hypersensitivity to Pyrazinamide and Related Amides

2000 ◽  
Vol 182 (19) ◽  
pp. 5479-5485 ◽  
Author(s):  
Helena I. M. Boshoff ◽  
Valerie Mizrahi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Intracellular Localization ◽  
Wild Type ◽  
Gene Encoding ◽  
Allelic Exchange ◽  
Mutant Strains ◽  
Established Role ◽  
Amidase Gene

ABSTRACT A pyrazinamidase (PZase)-deficient pncA mutant ofMycobacterium tuberculosis, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 μg/ml), in accordance with the well-established role ofpncA in the PZA susceptibility of M. tuberculosis (A. Scorpio and Y. Zhang, Nat. Med. 2:662–667, 1996). Integration of the pzaA gene encoding the major PZase/nicotinamidase from Mycobacterium smegmatis (H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809–5814, 1998) or the M. tuberculosis pncA gene into the pncAmutant complemented its PZase/nicotinamidase defect. In bothpzaA- and pncA-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. ThepzaA-complemented strain was hypersensitive to PZA (MIC, ≤10 μg/ml) and nicotinamide (MIC, ≥20 μg/ml) and was also sensitive to benzamide (MIC, 20 μg/ml), unlike the wild-type andpncA-complemented mutant strains, which were highly resistant to this amide (MIC, >500 μg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on M. smegmatis (MIC, 150 μg/ml in all cases) and rendered Escherichia colihypersensitive for growth at low pH.

scholarly journals Comparative reactions of recombinant papaya ringspot viruses with chimeric coat protein (CP) genes and wild-type viruses on CP-transgenic papaya

2001 ◽  
Vol 82 (11) ◽  
pp. 2827-2836 ◽  
Author(s):  
Chu-Hui Chiang ◽  
Ju-Jung Wang ◽  
Fuh-Jyh Jan ◽  
Shyi-Dong Yeh ◽  
Dennis Gonsalves
Keyword(s):  
Coat Protein ◽  
Sequence Similarity ◽  
Transcriptional Gene Silencing ◽  
Papaya Ringspot Virus ◽  
Wild Type ◽  
Coding Region ◽  
Transgenic Papaya ◽  
Cp Gene ◽  
Post Transcriptional Gene Silencing

Transgenic papaya cultivars SunUp and Rainbow express the coat protein (CP) gene of the mild mutant of papaya ringspot virus (PRSV) HA. Both cultivars are resistant to PRSV HA and other Hawaii isolates through homology-dependent resistance via post-transcriptional gene silencing. However, Rainbow, which is hemizygous for the CP gene, is susceptible to PRSV isolates from outside Hawaii, while the CP-homozygous SunUp is resistant to most isolates but susceptible to the YK isolate from Taiwan. To investigate the role of CP sequence similarity in overcoming the resistance of Rainbow, PRSV HA recombinants with various CP segments of the YK isolate were constructed and evaluated on Rainbow, SunUp and non-transgenic papaya. Non-transgenic papaya were severely infected by all recombinants, but Rainbow plants developed a variety of symptoms. On Rainbow, a recombinant with the entire CP gene of YK caused severe symptoms, while recombinants with only partial YK CP sequences produced a range of milder symptoms. Interestingly, a recombinant with a YK segment from the 5′ region of the CP gene caused very mild, transient symptoms, whereas recombinants with YK segments from the middle and 3′ parts of the CP gene caused prominent and lasting symptoms. SunUp was resistant to all but two recombinants, which contained the entire CP gene or the central and 3′-end regions of the CP gene and the 3′ non-coding region of YK, and the resulting symptoms were mild. It is concluded that the position of the heterologous sequences in the recombinants influences their pathogenicity on Rainbow.

The amoebae plate test implicates a paralogue of lpxB in the interaction of Legionella pneumophila with Acanthamoeba castellanii

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 167-182 ◽  
Author(s):  
Urs Albers ◽  
Katrin Reus ◽  
Howard A. Shuman ◽  
Hubert Hilbi
Keyword(s):  
Legionella Pneumophila ◽  
Lipid A ◽  
Sequence Similarity ◽  
Acanthamoeba Castellanii ◽  
Growth Defect ◽  
Wild Type ◽  
Intracellular Growth ◽  
Plate Test ◽  
Raw264.7 Macrophages ◽  
Mutant Strains

Legionella pneumophila is a bacterial parasite of freshwater amoebae which also grows in alveolar macrophages and thus causes the potentially fatal pneumonia Legionnaires' disease. Intracellular growth within amoebae and macrophages is mechanistically similar and requires the Icm/Dot type IV secretion system. This paper reports the development of an assay, the amoebae plate test (APT), to analyse growth of L. pneumophila wild-type and icm/dot mutant strains spotted on agar plates in the presence of Acanthamoeba castellanii. In the APT, wild-type L. pneumophila formed robust colonies even at high dilutions, icmT, -R, -P or dotB mutants failed to grow, and icmS or -G mutants were partially growth defective. The icmS or icmG mutant strains were used to screen an L. pneumophila chromosomal library for genes that suppress the growth defect in the presence of the amoebae. An icmS suppressor plasmid was isolated that harboured the icmS and flanking icm genes, indicating that this plasmid complements the intracellular growth defect of the mutant. In contrast, different icmG suppressor plasmids rendered the icmG mutant more cytotoxic for A. castellanii without enhancing intracellular multiplication in amoebae or RAW264.7 macrophages. Deletion of individual genes in the suppressor plasmids inserts identified lcs (Legionella cytotoxic suppressor) -A, -B, -C and -D as being required for enhanced cytotoxicity of an icmG mutant strain. The corresponding proteins show sequence similarity to hydrolases, NlpD-related metalloproteases, lipid A disaccharide synthases and ABC transporters, respectively. Overexpression of LcsC, a putative paralogue of the lipid A disaccharide synthase LpxB, increased cytotoxicity of an icmG mutant but not that of other icm/dot or rpoS mutant strains against A. castellanii. Based on sequence comparison and chromosomal location, lcsB and lcsC probably encode enzymes involved in cell wall maintenance and peptidoglycan metabolism. The APT established here may prove useful to identify other bacterial factors relevant for interactions with amoeba hosts.

LuxS modulates motility and secretion of extracellular protease in fish pathogen Vibrio harveyi

2021 ◽  
Author(s):  
yaqiu Zhang ◽  
Yiqing Deng ◽  
Juan Feng ◽  
Jianmei Hu ◽  
Haoxiang Chen ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Stationary Growth Phase ◽  
Extracellular Protease ◽  
Fish Pathogen ◽  
Wild Type ◽  
Stationary Growth ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Luxs Mutant

In this study, an in-frame deletion of the luxS gene was constructed to reveal the role of LuxS in the physiology and virulence of V. harveyi. The statistical analysis showed no significant differences in the growth ability, biofilm formation, antibiotic susceptibility, virulence by intraperitoneal injection, and the ability of V. harveyi to colonize the spleen and liver of the pearl gentian grouper between the wild-type (WT) and the luxS mutant. However, the deletion of luxS decreased the secretion of extracellular protease, while increased the ability of swimming and swarming. Simultaneously, a luxS-deleted mutant showed overproduction of lateral flagella, and an intact luxS complemented the defect. Since motility is flagella dependent, 16 of V. harveyi flagella biogenesis related genes were selected for further analysis. Based on quantitative real-time reverse transcription-PCR (qRT-PCR), the expression levels of these genes, including the polar flagella genes flaB, flhA, flhF, flhB, flhF, fliS, and flrA and the lateral flagella genes flgA, flgB, fliE, fliF, lafA, lafK, and motY, were significantly up-regulated in the ΔluxS: pMMB207 (ΔluxS+) strain as compared with the V. harveyi 345: pMMB207 (WT+) and C-ΔluxS strains during the early, mid-exponential, and stationary growth phase.

scholarly journals Role of CpALS4790 and CpALS0660 in Candida parapsilosis Virulence: Evidence from a Murine Model of Vaginal Candidiasis

2020 ◽  
Vol 6 (2) ◽  
pp. 86
Author(s):  
Marina Zoppo ◽  
Fabrizio Fiorentini ◽  
Cosmeri Rizzato ◽  
Mariagrazia Di Luca ◽  
Antonella Lupetti ◽  
...  
Keyword(s):  
Cell Wall ◽  
Murine Model ◽  
Type Strain ◽  
Wild Type Strain ◽  
Candida Parapsilosis ◽  
Vaginal Candidiasis ◽  
Phenotypic Characterization ◽  
Wild Type ◽  
Mutant Strains

The Candida parapsilosis genome encodes for five agglutinin-like sequence (Als) cell-wall glycoproteins involved in adhesion to biotic and abiotic surfaces. The work presented here is aimed at analyzing the role of the two still uncharacterized ALS genes in C. parapsilosis, CpALS4790 and CpALS0660, by the generation and characterization of CpALS4790 and CpALS066 single mutant strains. Phenotypic characterization showed that both mutant strains behaved as the parental wild type strain regarding growth rate in liquid/solid media supplemented with cell-wall perturbing agents, and in the ability to produce pseudohyphae. Interestingly, the ability of the CpALS0660 null mutant to adhere to human buccal epithelial cells (HBECs) was not altered when compared with the wild-type strain, whereas deletion of CpALS4790 led to a significant loss of the adhesion capability. RT-qPCR analysis performed on the mutant strains in co-incubation with HBECs did not highlight significant changes in the expression levels of others ALS genes. In vivo experiments in a murine model of vaginal candidiasis indicated a significant reduction in CFUs recovered from BALB/C mice infected with each mutant strain in comparison to those infected with the wild type strain, confirming the involvement of CpAls4790 and CpAls5600 proteins in C. parapsilosis vaginal candidiasis in mice.

scholarly journals Genetic and Genomic Insights into the Role of Benzoate-Catabolic Pathway Redundancy in Burkholderia xenovorans LB400

2006 ◽  
Vol 72 (1) ◽  
pp. 585-595 ◽  
Author(s):  
V. J. Denef ◽  
J. A. Klappenbach ◽  
M. A. Patrauchan ◽  
C. Florizone ◽  
J. L. M. Rodrigues ◽  
...  
Keyword(s):  
Polychlorinated Biphenyl ◽  
Gene Clusters ◽  
Large Chromosome ◽  
Wild Type ◽  
Burkholderia Xenovorans ◽  
Burkholderia Xenovorans Lb400 ◽  
Cell Assays ◽  
Aromatic Degradation ◽  
Proteomic Analyses

ABSTRACT Transcriptomic and proteomic analyses of Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB) degrader, have implicated growth substrate- and phase-dependent expression of three benzoate-catabolizing pathways: a catechol ortho cleavage (ben-cat) pathway and two benzoyl-coenzyme A pathways, encoded by gene clusters on the large chromosome (boxC ) and the megaplasmid (boxM ). To elucidate the significance of this apparent redundancy, we constructed mutants with deletions of the ben-cat pathway (the ΔbenABCD::kan mutant), the boxC pathway (the ΔboxABC ::kan mutant), and both pathways (the ΔbenABCDΔ boxABC ::kan mutant). All three mutants oxidized benzoate in resting-cell assays. However, the ΔbenABCD::kan and ΔbenABCD ΔboxABC ::kan mutants grew at reduced rates on benzoate and displayed increased lag phases. By contrast, growth on succinate, on 4-hydroxybenzoate, and on biphenyl was unaffected. Microarray and proteomic analyses revealed that cells of the ΔbenABCD::kan mutant growing on benzoate expressed both box pathways. Overall, these results indicate that all three pathways catabolize benzoate. Deletion of benABCD abolished the ability of LB400 to grow using 3-chlorobenzoate. None of the benzoate pathways could degrade 2- or 4-chlorobenzoate, indicating that the pathway redundancy does not directly contribute to LB400's PCB-degrading capacities. Finally, an extensive sigmaE-regulated oxidative stress response not present in wild-type LB400 grown on benzoate was detected in these deletion mutants, supporting our earlier suggestion that the box pathways are preferentially active under reduced oxygen tension. Our data further substantiate the expansive network of tightly interconnected and complexly regulated aromatic degradation pathways in LB400.

scholarly journals Role of FAD-I in Fusobacterial Interspecies Interaction and Biofilm Formation

2020 ◽  
Vol 8 (1) ◽  
pp. 70 ◽  
Author(s):  
Bhumika Shokeen ◽  
Jane Park ◽  
Emily Duong ◽  
Sonam Rambhia ◽  
Manash Paul ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Fusobacterium Nucleatum ◽  
Interspecies Interaction ◽  
Wild Type ◽  
Oral Epithelial Cells ◽  
Small Proteins ◽  
Mutant Strains ◽  
Oral Epithelial ◽  
Wild Type Parent

RadD, a major adhesin of oral fusobacteria, is part of a four-gene operon encoding the small lipoprotein FAD-I and two currently uncharacterized small proteins encoded by the rapA and rapB genes. Previously, we described a role for FAD-I in the induction of human B-defensin 2 (hBD2) upon contact with oral epithelial cells. Here, we investigated potential roles for fad-I, rapA, and rapB in interspecies interaction and biofilm formation. Gene inactivation mutants were generated for each of these genes in the nucleatum and polymorphum subspecies of Fusobacterium nucleatum and characterized for their adherence to partner species, biofilm formation, and operon transcription. Binding to Streptococcus gordonii was increased in all mutant strains with Δfad-I having the most significant effect. This increased adherence was directly proportional to elevated radD transcript levels and resulted in significantly different architecture and height of the biofilms formed by Δfad-I and S. gordonii compared to the wild-type parent. In conclusion, FAD-I is important for fusobacterial interspecies interaction as its lack leads to increased production of the RadD adhesin suggesting a role of FAD-I in its regulation. This regulatory effect does not require the presence of functional RadD.

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