scholarly journals The pnhA Gene of Pasteurella multocida Encodes a Dinucleoside Oligophosphate Pyrophosphatase Member of the Nudix Hydrolase Superfamily

2005 ◽  
Vol 187 (16) ◽  
pp. 5809-5817 ◽  
Author(s):  
Tonia Urick ◽  
Chien I-Chang ◽  
Ellen Arena ◽  
WenLian Xu ◽  
Maurice J. Bessman ◽  
...  
Keyword(s):  
Type Strain ◽  
Pasteurella Multocida ◽  
Wild Type Strain ◽  
Divalent Metal ◽  
Nudix Hydrolase ◽  
Alkaline Ph ◽  
Wild Type ◽  
Optimal Activity ◽  
Diadenosine Pentaphosphate ◽  
Hydrolysis Of

ABSTRACT The pnhA gene of Pasteurella multocida encodes PnhA, which is a member of the Nudix hydrolase subfamily of dinucleoside oligophosphate pyrophosphatases. PnhA hydrolyzes diadenosine tetra-, penta-, and hexaphosphates with a preference for diadenosine pentaphosphate, from which it forms ATP and ADP. PnhA requires a divalent metal cation, Mg2+ or Mn2+, and prefers an alkaline pH of 8 for optimal activity. A P. multocida strain that lacked a functional pnhA gene, ACP13, was constructed to further characterize the function of PnhA. The cellular size of ACP13 was found to be 60% less than that of wild-type P. multocida, but the growth rate of ACP13 and its sensitivity to heat shock conditions were similar to those of the wild type, and the wild-type cell size was restored in the presence of a functional pnhA gene. Wild-type and ACP13 strains were tested for virulence by using the chicken embryo lethality model, and ACP13 was found to be up to 1,000-fold less virulent than the wild-type strain. This is the first study to use an animal model in assessing the virulence of a bacterial strain that lacked a dinucleoside oligophosphate pyrophosphatase and suggests that the pyrophosphatase PnhA, catalyzing the hydrolysis of diadenosine pentaphosphates, may also play a role in facilitating P. multocida pathogenicity in the host.

scholarly journals Role of aspartate ammonia-lyase in Pasteurella multocida

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Zui Wang ◽  
Li Li ◽  
Peng Liu ◽  
Chen Wang ◽  
Qin Lu ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Bacterial Growth ◽  
Type Strain ◽  
Pasteurella Multocida ◽  
Wild Type Strain ◽  
Luria Bertani ◽  
Economic Losses ◽  
Wild Type ◽  
Mueller Hinton ◽  
Significant Difference

Abstract Background Pasteurella multocida is responsible for a highly infectious and contagious disease in birds, leading to heavy economic losses in the chicken industry. However, the pathogenesis of this disease is poorly understood. We recently identified an aspartate ammonia-lyase (aspA) in P. multocida that was significantly upregulated under iron-restricted conditions, the protein of which could effectively protect chicken flocks against P. multocida. However, the functions of this gene remain unclear. In the present study, we constructed aspA mutant strain △aspA::kan and complementary strain C△aspA::kan to investigate the function of aspA in detail. Result Deletion of the aspA gene in P. multocida resulted in a significant reduction in bacterial growth in LB (Luria-Bertani) and MH (Mueller-Hinton) media, which was rescued by supplementation with 20 mM fumarate. The mutant strain △aspA::kan showed significantly growth defects in anaerobic conditions and acid medium, compared with the wild-type strain. Moreover, growth of △aspA::kan was more seriously impaired than that of the wild-type strain under iron-restricted conditions, and this growth recovered after supplementation with iron ions. AspA transcription was negatively regulated by iron conditions, as demonstrated by quantitative reverse transcription-polymerase chain reaction. Although competitive index assay showed the wild-type strain outcompetes the aspA mutant strain and △aspA::kan was significantly more efficient at producing biofilms than the wild-type strain, there was no significant difference in virulence between the mutant and the wild-type strains. Conclusion These results demonstrate that aspA is required for bacterial growth in complex medium, and under anaerobic, acid, and iron-limited conditions.

scholarly journals Dual Functions of Streptococcus salivarius Urease

2000 ◽  
Vol 182 (16) ◽  
pp. 4667-4669 ◽  
Author(s):  
Yi-Ywan M. Chen ◽  
Cheryl A. Weaver ◽  
Robert A. Burne
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Streptococcus Salivarius ◽  
Wild Type ◽  
Allelic Exchange ◽  
Hydrolysis Of ◽  
Dependent Pathway ◽  
Dual Functions

ABSTRACT A urease-deficient derivative of Streptococcus salivarius 57.I was constructed by allelic exchange at theureC locus. The wild-type strain was protected against acid killing through hydrolysis of physiologically relevant concentrations of urea, whereas the mutant was not. Also, S. salivariuscould use urea as a source of nitrogen for growth exclusively through a urease-dependent pathway.

scholarly journals The lipopolysaccharide outer core transferase genes pcgD and hptE contribute differently to the virulence of Pasteurella multocida in ducks

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Xinxin Zhao ◽  
Hui Shen ◽  
Sheng Liang ◽  
Dekang Zhu ◽  
Mingshu Wang ◽  
...  
Keyword(s):  
Membrane Permeability ◽  
Outer Membrane ◽  
Type Strain ◽  
Pasteurella Multocida ◽  
Wild Type Strain ◽  
Outer Core ◽  
High Dose ◽  
Wild Type ◽  
Virulence Attenuation ◽  
Outer Membrane Permeability

AbstractFowl cholera caused by Pasteurella multocida exerts a massive economic burden on the poultry industry. Lipopolysaccharide (LPS) is essential for the growth of P. multocida genotype L1 strains in chickens and specific truncations to the full length LPS structure can attenuate bacterial virulence. Here we further dissected the roles of the outer core transferase genes pcgD and hptE in bacterial resistance to duck serum, outer membrane permeability and virulence in ducks. Two P. multocida mutants, ΔpcgD and ΔhptE, were constructed, and silver staining confirmed that they all produced truncated LPS profiles. Inactivation of pcgD or hptE did not affect bacterial susceptibility to duck serum and outer membrane permeability but resulted in attenuated virulence in ducks to some extent. After high-dose inoculation, ΔpcgD showed remarkably reduced colonization levels in the blood and spleen but not in the lung and liver and caused decreased injuries in the spleen and liver compared with the wild-type strain. In contrast, the ΔhptE loads declined only in the blood, and ΔhptE infection caused decreased splenic lesions but also induced severe hepatic lesions. Furthermore, compared with the wild-type strain, ΔpcgD was significantly attenuated upon oral or intramuscular challenge, whereas ΔhptE exhibited reduced virulence only upon oral infection. Therefore, the pcgD deletion caused greater virulence attenuation in ducks, indicating the critical role of pcgD in P. multocida infection establishment and survival.

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

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
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.

scholarly journals The role of Zur-regulated lipoprotein A in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles in Acinetobacter baumannii

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.

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

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.

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

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.

scholarly journals Mutations of folC cause increased susceptibility to sulfamethoxazole in Mycobacterium tuberculosis

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.

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

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.

scholarly journals Homology-Dependent Silencing of the SC3 Gene in Schizophyllum commune

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 589-596 ◽  
Author(s):  
Theo A Schuurs ◽  
Eveline A M Schaeffer ◽  
Joseph G H Wessels
Keyword(s):  
Gene Silencing ◽  
Genomic Dna ◽  
Type Strain ◽  
Cytosine Methylation ◽  
Wild Type Strain ◽  
Schizophyllum Commune ◽  
Southern Analysis ◽  
Transcriptional Level ◽  
Wild Type

After introduction of extra copies of the SC3 hydrophobin gene into a wild-type strain of Schizophyllum commune, gene silencing was observed acting on both endogenous and introduced SC3 genes in primary vegetative transformants. Nuclear run-on experiments indicated that silencing acted at the transcriptional level. Southern analysis revealed that cytosine methylation of genomic DNA occurred. Moreover, SC3 silencing was suppressed by exposure to 5-azacytidine during growth. After growth of SC3-suppressed colonies from homogenized mycelium or from colonies stored at 4°, SC3 transcription was restored. However, after prolonged growth SC3 silencing was again observed. Introduction of a promoterless SC3 fragment into wild type gave less SC3 silencing.

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