scholarly journals Involvement of H-NS in Acinetobacter baumannii’s Natural Transformation

2021 ◽  
Author(s):  
Casin Le ◽  
Camila Pimentel ◽  
Marisel Romina Tuttobene ◽  
Tomás Subils ◽  
Jenny Escalante ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Wild Type Strain ◽  
Natural Transformation ◽  
Regulatory Elements ◽  
Wild Type ◽  
Regulation Of Expression ◽  
Expression Levels ◽  
Expression Of Genes ◽  
Foreign Dna

Most Acinetobacter baumannii strains are naturally competent. Although some information is available about factors that enhance or reduce the frequency of transformation of this bacterium, the regulatory elements and mechanisms are barely understood. In this article, we describe studies on the role of H-NS in the regulation of expression of genes related to natural competency and the ability to uptake foreign DNA. The expression levels of the natural transformation-related genes pilA, pilT, pilQ, comEA, comEC, comF, and drpA were significantly increased in a Δhns derivative of Acinetobacter baumannii A118. Complementation of the mutant with a recombinant plasmid harboring hns restored expression levels of six of these genes (pilT remained expressed at high levels) to those of the wild-type strain. The transformation frequency of the A. baumannii A118 Δhns strain was significantly higher than that of the wild-type. Similar, albeit not identical, effects occurred when hns was deleted from the hypervirulent A. baumannii AB5075 strain. Reduction of gene expression in a few cases was not as pronounced as to reach wild-type levels, and expression of comEA was enhanced further. In conclusion, the expression of all seven transformation-related genes was enhanced after deleting hns in A. baumannii A118 and AB5075, and these modifications are accompanied by an increase in the cells’ transformability. The results demonstrate a role of H-NS in A. baumannii’s natural competence.

scholarly journals Involvement of the Histone-Like Nucleoid Structuring Protein (H-NS) in Acinetobacter baumannii’s Natural Transformation

Pathogens ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1083
Author(s):  
Casin Le ◽  
Camila Pimentel ◽  
Marisel R. Tuttobene ◽  
Tomás Subils ◽  
Jenny Escalante ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Natural Transformation ◽  
Regulatory Elements ◽  
Transformation Frequency ◽  
Wild Type ◽  
Natural Competence ◽  
Expression Levels ◽  
Expression Of Genes ◽  
Foreign Dna

Most Acinetobacter baumannii strains are naturally competent. Although some information is available about factors that enhance or reduce the frequency of the transformation of this bacterium, the regulatory elements and mechanisms are barely understood. In this article, we describe studies on the role of the histone-like nucleoid structuring protein, H-NS, in the regulation of the expression of genes related to natural competency and the ability to uptake foreign DNA. The expression levels of the natural transformation-related genes pilA, pilT, pilQ, comEA, comEC, comF, and drpA significantly increased in a Δhns derivative of A. baumannii A118. The complementation of the mutant with a recombinant plasmid harboring hns restored the expression levels of six of these genes (pilT remained expressed at high levels) to those of the wild-type strain. The transformation frequency of the A. baumannii A118 Δhns strain was significantly higher than that of the wild-type. Similar, albeit not identical, there were consequences when hns was deleted from the hypervirulent A. baumannii AB5075 strain. In the AB5075 complemented strain, the reduction in gene expression in a few cases was not so pronounced that it reached wild-type levels, and the expression of comEA was enhanced further. In conclusion, the expression of all seven transformation-related genes was enhanced after deleting hns in A. baumannii A118 and AB5075, and these modifications were accompanied by an increase in the cells’ transformability. The results highlight a role of H-NS in A. baumannii’s natural competence.

scholarly journals Interplay between meropenem and human serum albumin on expression of carbapenem resistance genes and natural competence in Acinetobacter baumannii

2021 ◽  
Author(s):  
Casin Le ◽  
Camila Pimentel ◽  
Marisel R. Tuttobene ◽  
Tomas Subils ◽  
Brent Nishimura ◽  
...  
Keyword(s):  
Human Serum ◽  
Acinetobacter Baumannii ◽  
Resistance Genes ◽  
Natural Transformation ◽  
Expression Levels ◽  
Carbapenem Resistant ◽  
Synergistic Enhancement ◽  
Expression Of Genes ◽  
Foreign Dna ◽  
Accelerate Evolution

Acinetobacter baumannii A118, a mostly susceptible strain and AB5075, carbapenem-resistant, were cultured in Lysogeny broth (LB) or LB with different supplements: 3.5% human serum albumin (HSA), human serum (HS), meropenem, or meropenem plus 3.5% HSA. Natural transformation levels were enhanced in A. baumannii A118 and AB5075 cultured in medium supplemented with 3.5% HSA. Addition of meropenem plus 3.5% HSA caused synergistic enhancement of natural transformation in A. baumannii A118. Medium containing 3.5% HSA or meropenem enhanced the expression levels of the competence and type IV pilus associated genes. The combination meropenem plus 3.5% HSA produced a synergistic enhancement in the expression levels of many of these genes. The addition of HS, which has a high content of HSA, was also an inducer of these genes. Cultures grown in medium supplemented with HS or 3.5% HSA also affected resistance genes, which were expressed at higher or lower levels depending on the modification required to enhance resistance. The inducing or repressing activity of these modulators also occurred in three more carbapenem-resistant strains tested. An exception was the A. baumannii AMA16 bla NDM-1 gene, which was repressed in the presence of 3.5% HSA. In conclusion, HSA produces an enhancement of natural transformation and a modification in expression levels of competence genes and antibiotic resistance. Furthermore, when HSA is combined with carbapenems, which may increase the stress response, the expression of genes involved in natural competence is increased in A. baumannii . This process may favor the acquisition of foreign DNA and accelerate evolution. Importance Acinetobacter baumannii causes a variety of nosocomial- and community-infections that are usually resistant to multiple antimicrobial agents. As new strains acquire more resistance genes, these infections become more difficult to treat and mortality can reach up to 39%. The high genomic plasticity exhibited by A. baumannii must be the consequence of numerous mechanisms that include acquiring foreign DNA and recombination. Here, we describe the ability of A. baumannii to induce competence genes when exposed to environments that resemble those found in the human body during untreated infection or after administration of carbapenems. In this latter scenario expression of genes related to resistance also modify their expression levels such that resistance is increased. The contributions of this article are two-fold. Firstly, when A. baumannii is exposed to products present during infection, it responds, augmenting the ability to capture DNA and accelerate evolution. Secondly, in those conditions, the bacterium also modifies the expression of resistance genes to increase its resistance levels. In summary, recognition of substances that are naturally (e.g., HSA) or artificially (treatment with carbapenems) induces A. baumannii to enhance expression of resistance determinants and genes regulating competence.

scholarly journals Transcriptomics-Aided Dissection of the Intracellular and Extracellular Roles of Microcystin in Microcystis aeruginosa PCC 7806

2014 ◽  
Vol 81 (2) ◽  
pp. 544-554 ◽  
Author(s):  
A. Katharina Makower ◽  
J. Merijn Schuurmans ◽  
Detlef Groth ◽  
Yvonne Zilliges ◽  
Hans C. P. Matthijs ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Wild Type Strain ◽  
Carbon Limitation ◽  
Light Conditions ◽  
Wild Type ◽  
Low Light ◽  
Content Type ◽  
Expression Of Genes ◽  
Polyketide Synthase Gene

ABSTRACTRecent studies have provided evidence for both intracellular and extracellular roles of the potent hepatotoxin microcystin (MC) in the bloom-forming cyanobacteriumMicrocystis. Here, we surveyed transcriptomes of the wild-type strainM. aeruginosaPCC 7806 and the microcystin-deficient ΔmcyBmutant under low light conditions with and without the addition of external MC of the LR variant (MC-LR). Transcriptomic data acquired by microarray and quantitative PCR revealed substantial differences in the relative expression of genes of the central intermediary metabolism, photosynthesis, and energy metabolism. In particular, the data provide evidence for a lower photosystem I (PSI)-to-photosystem II (PSII) ratio and a more pronounced carbon limitation in the microcystin-deficient mutant. Interestingly, only 6% of the transcriptional differences could be complemented by external microcystin-LR addition. This MC signaling effect was seen exclusively for genes of the secondary metabolism category. The orphan polyketide synthase gene cluster IPF38-51 was specifically downregulated in response to external MC-LR under low light. Our data suggest a hierarchical and light-dependent cross talk of secondary metabolites and support both an intracellular and an extracellular role of MC inMicrocystis.

scholarly journals ClpC acts as a negative regulator of competence in Streptococcus thermophilus

Microbiology ◽  
2011 ◽  
Vol 157 (6) ◽  
pp. 1676-1684 ◽  
Author(s):  
Truls Johan Biørnstad ◽  
Leiv Sigve Håvarstein
Keyword(s):  
Sigma Factor ◽  
Wild Type Strain ◽  
Natural Transformation ◽  
Streptococcus Thermophilus ◽  
Negative Regulator ◽  
Wild Type ◽  
Environmental Stimulus ◽  
Mutant Strains ◽  
Low Levels

The alternative sigma factor ComX is a key regulator of natural transformation in members of the genus Streptococcus. ComX controls expression of the late competence genes, which are essential for DNA binding, uptake and recombination. In Streptococcus pneumoniae, it has been demonstrated that ComX is degraded by ClpEP at the end of the competence period. In the present study we show that a different Clp protease complex, ClpCP, contributes to ComX degradation in Streptococcus thermophilus. Mutant strains lacking the ClpC chaperone displayed significantly increased transformability compared with the wild-type strain under conditions where ComX was expressed at relatively low levels. At higher expression levels, ClpCP appears to become saturated and unable to prevent the accumulation of ComX. Together, our results suggest that the role of ClpC is to mediate degradation of ComX when the sigma factor is produced in low amounts, i.e. when the environmental stimulus promoting competence development is weak. This would prevent S. thermophilus from developing the competent state at an inappropriate time and/or place.

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 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 Enhanced production of Clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain

2021 ◽  
Author(s):  
Chang-Hun Shin ◽  
Hang Soo Cho ◽  
Hyung-Jin Won ◽  
Ho Jeong Kwon ◽  
Chan-Wha Kim ◽  
...  
Keyword(s):  
Clavulanic Acid ◽  
Wild Type Strain ◽  
Random Mutagenesis ◽  
Streptomyces Clavuligerus ◽  
Wild Type ◽  
Mutant Selection ◽  
Enhanced Production ◽  
Industrial Strains ◽  
Glycerol Utilization

Abstract Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3 per cent (5.21 ± 0.26 g/L) in a flask culture and 17.4 per cent (6.11 ± 0.36 g/L) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

Neurotensin Enhances Locomotor Activity and Arousal and Inhibits Melanin-Concentrating Hormone Signaling

2019 ◽  
Vol 110 (1-2) ◽  
pp. 35-49 ◽  
Author(s):  
Talia Levitas-Djerbi ◽  
Dana Sagi ◽  
Ilana Lebenthal-Loinger ◽  
Tali Lerer-Goldshtein ◽  
Lior Appelbaum
Keyword(s):  
Locomotor Activity ◽  
Hormone Receptor ◽  
Behavioral Response ◽  
Hormone Signaling ◽  
Wild Type ◽  
Expression Levels ◽  
Physiological Processes ◽  
Melanin Concentrating Hormone ◽  
Behavioral Assays

Background: Hypothalamic neurotensin (Nts)-secreting neurons regulate fundamental physiological processes including metabolism and feeding. However, the role of Nts in modulation of locomotor activity, sleep, and arousal is unclear. We previously identified and characterized Nts neurons in the zebrafish hypothalamus. Materials and Methods: In order to study the role of Nts, nts mutant (nts–/–), and overexpressing zebrafish were generated. Results: The expression of both nts mRNA and Nts protein was reduced during the night in wild-type zebrafish. Behavioral assays revealed that locomotor activity was decreased during both day and night, while sleep was increased exclusively during the nighttime in nts–/– larvae. Likewise, inducible overexpression of Nts increased arousal in hsp70:Gal4/uas:Nts larvae. Furthermore, the behavioral response to light-to-dark transitions was reduced in nts–/– larvae. In order to elucidate potential contenders that may mediate Nts action on these behaviors, we profiled the transcriptome of 6 dpf nts–/– larvae. Among other genes, the expression levels of melanin-concentrating hormone receptor 1b were increased in nts–/– larvae. Furthermore, a portion of promelanin-concentrating hormone 1 (pmch1) and pmch2 neurons expressed the nts receptor. In addition, expression of the the two zebrafish melanin-concentrating hormone (Mch) orthologs, Mch1 and Mch2, was increased in nts–/– larvae. Conclusion: These results show that the Nts and Mch systems interact and modulate locomotor activity and arousal.

scholarly journals Autoproteolysis of YscU of Yersinia pseudotuberculosis Is Important for Regulation of Expression and Secretion of Yop Proteins

2009 ◽  
Vol 191 (13) ◽  
pp. 4259-4267 ◽  
Author(s):  
Ann-Catrin Björnfot ◽  
Moa Lavander ◽  
Åke Forsberg ◽  
Hans Wolf-Watz
Keyword(s):  
Type Iii Secretion ◽  
Calcium Concentration ◽  
Wild Type Strain ◽  
Yersinia Pseudotuberculosis ◽  
Secretion System ◽  
Wild Type ◽  
Regulation Of Expression ◽  
Significant Difference ◽  
In The Wild ◽  
Needle Protein

ABSTRACT YscU of Yersinia can be autoproteolysed to generate a 10-kDa C-terminal polypeptide designated YscUCC. Autoproteolysis occurs at the conserved N↓PTH motif of YscU. The specific in-cis-generated point mutants N263A and P264A were found to be defective in proteolysis. Both mutants expressed and secreted Yop proteins (Yops) in calcium-containing medium (+Ca2+ conditions) and calcium-depleted medium (−Ca2+ conditions). The level of Yop and LcrV secretion by the N263A mutant was about 20% that of the wild-type strain, but there was no significant difference in the ratio of the different secreted Yops, including LcrV. The N263A mutant secreted LcrQ regardless of the calcium concentration in the medium, corroborating the observation that Yops were expressed and secreted in Ca2+-containing medium by the mutant. YscF, the type III secretion system (T3SS) needle protein, was secreted at elevated levels by the mutant compared to the wild type when bacteria were grown under +Ca2+ conditions. YscF secretion was induced in the mutant, as well as in the wild type, when the bacteria were incubated under −Ca2+ conditions, although the mutant secreted smaller amounts of YscF. The N263A mutant was cytotoxic for HeLa cells, demonstrating that the T3SS-mediated delivery of effectors was functional. We suggest that YscU blocks Yop release and that autoproteolysis is required to relieve this block.

scholarly journals Role Of Selected Fimbrial Adheins In Pathogenesis Of Acid-Induced Eneterohemorrhagic Escherichia Coli 0157:H7

2021 ◽  
Author(s):  
Shahnaz Haque
Keyword(s):  
Fatty Acid ◽  
Type Strain ◽  
Wild Type Strain ◽  
Short Chain Fatty Acid ◽  
Acid Stress ◽  
Chain Fatty Acid ◽  
Short Chain ◽  
Wild Type ◽  
Food Borne

Enterohemorrhagic Escherichia coli (EHEC) 0157:H7 is a food-borne pathogen that causes hemolytic uremic syndrome and hemorrhagic colitis. The mechanisms underlying the adhesion of EHEC 0157:H7 to intestinal epithelial cells are not well understood. Like other food-borne pathogens, ECEC 0157:H7 must survive the acid stress of the gastric juice in the stomach and short chain fatty acid in the intestine in order to colonize the large intestine. We have found that acid stress and short chain fatty acid stress significantly enhance host-adhesion of EHEC 0157:H7 and also upregulates expression of EHEC fimbrial genes, lpfA1, lpfA2 and yagZ, as demonstrated by our DNA microarray. We now report that disruption of the yagZ (also known as the E. coli common pilus A) gene results in loss of the acid-induced and short chain fatty acid-induced adhesion increase seen for the wild type strain. When the yagZ mutant is complemented with yagZ, the sress-induced and short chain fatty acid-induced adhesion increase seen for the wild type strain. When the yagZ mutant is complemented with yagZ, the stress-induced adhesion pehnotype is restored, confirming the role of yagZ in the acid as well as short chain fatty acid induced adhesion to HEp-2 cells. On the other hand, neither disruption in the long polar fimbria genes lpfA1 or lpfA2 in the wild type showed any effect in adherence to HEp-2 cells; rather displaying a hyperadherant phenotype to HEp-2 cells after acid-induced or short chain fatty acid-induced stress. The results also indicate that acid or short chain fatty acid stress, which is a part of the host's natural defense mechanism against pathogens, may regulate virulence factors resulting in enhanced bacteria-host attachment during colonization in the human or bovine host.

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