| 1. |
- Akar, Roya, et al.
(författare)
-
Regulation of the general stress response sigma factor σT by Lon-mediated proteolysis
- 2023
-
Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 205:11
-
Tidskriftsartikel (refereegranskat)abstract
- The Lon protease is widely conserved in both prokaryotic and eukaryotic organisms and fulfills important regulatory functions. Nevertheless, the number of identified Lon substrates is limited in most organisms, and the precise role of Lon in regulating these proteins is poorly understood. Here, we describe the α-proteobacterial general stress response sigma factor σT as a novel Lon substrate in Caulobacter crescentus. Based on previously published quantitative proteomics data, we find σT to be a promising putative Lon substrate and confirm a direct role of Lon in degrading σT. We show that Lon contributes to the downregulation of σT abundance under optimal conditions and during recovery from sucrose-induced osmotic stress. Furthermore, the presence of the Lon activity regulator LarA enhances Lon-mediated degradation of σT in vitro and reduces σT levels in vivo indicating a role of LarA in modulating Lon-mediated degradation of σT. Together, our results highlight the importance of Lon during the recovery phase following stress exposure by adjusting the concentrations of critical regulators of stress responses.
|
|
| 2. |
- Amon, Jeremy D., et al.
(författare)
-
SwsB and SafA Are Required for CwlJ-Dependent Spore Germination in Bacillus subtilis
- 2020
-
Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 202:6
-
Tidskriftsartikel (refereegranskat)abstract
- When Bacillus subtilis spores detect nutrients, they exit dormancy through the processes of germination and outgrowth. A key step in germination is the activation of two functionally redundant cell wall hydrolases (SleB and CwlJ) that degrade the specialized cortex peptidoglycan that surrounds the spore. How these enzymes are regulated remains poorly understood. To identify additional factors that affect their activity, we used transposon sequencing to screen for synthetic germination defects in spores lacking SleB or CwlJ. Other than the previously characterized protein YpeB, no additional factors were found to be specifically required for SleB activity. In contrast, our screen identified SafA and YlxY (renamed SwsB) in addition to the known factors GerQ and CotE as proteins required for CwlJ function. SafA is a member of the spore's proteinaceous coat and we show that, like GerQ and CotE, it is required for accumulation and retention of CwlJ in the dormant spore. SwsB is broadly conserved among spore formers, and we show that it is required for CwlJ to efficiently degrade the cortex during germination. Intriguingly, SwsB resembles polysaccharide deacetylases, and its putative catalytic residues are required for its role in germination. However, we find no chemical signature of its activity on the spore cortex or in vitro. While the precise, mechanistic role of SwsB remains unknown, we explore and discuss potential activities. IMPORTANCE Spore formation in Bacillus subtilis has been studied for over half a century, and virtually every step in this developmental process has been characterized in molecular detail. In contrast, how spores exit dormancy remains less well understood. A key step in germination is the degradation of the specialized cell wall surrounding the spore called the cortex. Two enzymes (SleB and CwlJ) specifically target this protective layer, but how they are regulated and whether additional factors promote their activity are unknown. Here, we identified the coat protein SafA and a conserved but uncharacterized protein YlxY as additional factors required for CwlJ-dependent degradation of the cortex. Our analysis provides a more complete picture of this essential step in the exit from dormancy.
|
|
| 3. |
- Bueno, Emilio, et al.
(författare)
-
Genetic Dissection of the Fermentative and Respiratory Contributions Supporting Vibrio cholerae Hypoxic Growth
- 2020
-
Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 202:24
-
Tidskriftsartikel (refereegranskat)abstract
- Both fermentative and respiratory processes contribute to bacterial metabolic adaptations to low oxygen tension (hypoxia). In the absence of O-2 as a respiratory electron sink, many bacteria utilize alternative electron acceptors, such as nitrate (NO3-). During canonical NO3- respiration, NO3- is reduced in a stepwise manner to N-2 by a dedicated set of reductases. Vibrio cholerae, the etiological agent of cholera, requires only a single periplasmic NO3- reductase (NapA) to undergo NO3- respiration, suggesting that the pathogen possesses a noncanonical NO3- respiratory chain. In this study, we used complementary transposon-based screens to identify genetic determinants of general hypoxic growth and NO3- respiration in V. cholerae. We found that while the V. cholerae NO3- respiratory chain is primarily composed of homologues of established NO3- respiratory genes, it also includes components previously unlinked to this process, such as the Na+-NADH dehydrogenase Nqr. The ethanol-generating enzyme AdhE was shown to be the principal fermentative branch required during hypoxic growth in V. cholerae. Relative to single adhE or napA mutant strains, a V. cholerae strain lacking both genes exhibited severely impaired hypoxic growth in vitro and in vivo. Our findings reveal the genetic basis of a specific interaction between disparate energy production pathways that supports pathogen fitness under shifting conditions. Such metabolic specializations in V. cholerae and other pathogens are potential targets for antimicrobial interventions.IMPORTANCE Bacteria reprogram their metabolism in environments with low oxygen levels (hypoxia). Typically, this occurs via regulation of two major, but largely independent, metabolic pathways: fermentation and respiration. In this study, we found that the diarrheal pathogen Vibrio cholerae has a respiratory chain for NO3- that consists largely of components found in other NO3- respiratory systems but also contains several proteins not previously linked to this process. Both AdhE-dependent fermentation and NO3- respiration were required for efficient pathogen growth under both laboratory conditions and in an animal infection model. These observations provide a specific example of fermentative respiratory interactions and identify metabolic vulnerabilities that may be targetable for new antimicrobial agents in V. cholerae and related pathogens.
|
|
| 4. |
- Bukowska-Faniband, Ewa, et al.
(författare)
-
Studies on Bd0934 and Bd3507, two secreted nucleases from bdellovibrio bacteriovorus, reveal sequential release of nucleases during the predatory cycle
- 2020
-
Ingår i: Journal of Bacteriology. - 0021-9193. ; 202:18
-
Tidskriftsartikel (refereegranskat)abstract
- Bdellovibrio bacteriovorus is an obligate predatory bacterium that invades and kills a broad range of Gram-negative prey cells, including human pathogens. Its potential therapeutic application has been the subject of increased research interest in recent years. However, an improved understanding of the fundamental molecular aspects of the predatory life cycle is crucial for developing this bacterium as a “living antibiotic.” During intracellular growth, B. bacteriovorus secretes an arsenal of hydrolases, which digest the content of the host cell to provide growth nutrients for the predator, e.g., prey DNA is completely degraded by the nucleases. Here, we have, on a genetic and molecular level, characterized two secreted DNases from B. bacteriovorus, Bd0934 and Bd3507, and determined the temporal expression profile of other putative secreted nucleases. We conclude that Bd0934 and Bd3507 are likely a part of the predatosome but are not essential for the predation, host-independent growth, prey biofilm degradation, and self-biofilm formation. The detailed temporal expression analysis of genes encoding secreted nucleases revealed that these enzymes are produced in a sequential orchestrated manner. This work contributes to our understanding of the sequential breakdown of the prey nucleic acid by the nucleases secreted during the predatory life cycle of B. bacteriovorus. IMPORTANCE Antibiotic resistance is a major global concern with few available new means to combat it. From a therapeutic perspective, predatory bacteria constitute an interesting tool. They not only eliminate the pathogen but also reduce the overall pool of antibiotic resistance genes through secretion of nucleases and complete degradation of exogenous DNA. Molecular knowledge of how these secreted DNases act will give us further insight into how antibiotic resistance, and the spread thereof, can be limited through the action of predatory bacteria.
|
|
| 5. |
- de Oliveira, Ana Henriques, et al.
(författare)
-
Listeria monocytogenes requires the RsbX protein to prevent SigB-activation under non-stressed conditions
- 2022
-
Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 204:1
-
Tidskriftsartikel (refereegranskat)abstract
- The survival of microbial cells under changing environmental conditions requires an efficient reprogramming of transcription, often mediated by alternative sigma factors. The Gram-positive human pathogen Listeria monocytogenes senses and responds to environmental stress mainly through the alternative sigma factor σB (SigB), which controls expression of the general stress response regulon. SigB activation is achieved through a complex series of phosphorylation/dephosphorylation events culminating in the release of SigB from its anti-sigma factor RsbW. At the top of the signal transduction pathway lies a large multi-protein complex known as the stressosome that is believed to act as a sensory hub for stresses. Following signal detection, stressosome proteins become phosphorylated. Resetting of the stressosome is hypothesized to be exerted by a putative phosphatase, RsbX, which presumably removes phosphate groups from stressosome proteins post-stress.We addressed the role of the RsbX protein in modulating the activity of the stressosome and consequently regulating SigB activity in L. monocytogenes. We show that RsbX is required to reduce SigB activation/levels under non-stress conditions and that it is required for appropriate SigB mediated stress-adaptation. A strain lacking RsbX displayed impaired motility and biofilm formation, but also an increased survival at low pH. Our results could suggest that absence of RsbX alter the multi-protein composition of the stressosome without dramatically affecting its phosphorylation status. Overall the data show that RsbX plays a critical role in modulating the signal transduction pathway by blocking SigB activation under non-stressed conditions.
|
|
| 6. |
- Guerreiro, Duarte N., et al.
(författare)
-
Mild Stress Conditions during Laboratory Culture Promote the Proliferation of Mutations That Negatively Affect Sigma B Activity in Listeria monocytogenes
- 2020
-
Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 202:9
-
Tidskriftsartikel (refereegranskat)abstract
- In Listeria monocytogenes, the full details of how stress signals are integrated into the σB regulatory pathway are not yet available. To help shed light on this question, we investigated a collection of transposon mutants that were predicted to have compromised activity of the alternative sigma factor B (σB). These mutants were tested for acid tolerance, a trait that is known to be under σB regulation, and they were found to display increased acid sensitivity, similar to a mutant lacking σB (ΔsigB). The transposon insertions were confirmed by whole-genome sequencing, but in each case, the strains were also found to carry a frameshift mutation in the sigB operon. The changes were predicted to result in premature stop codons, with negative consequences for σB activation, independently of the transposon location. Reduced σB activation in these mutants was confirmed. Growth measurements under conditions similar to those used during the construction of the transposon library revealed that the frameshifted sigB operon alleles conferred a growth advantage at higher temperatures, during late exponential phase. Mixed-culture experiments at 42°C demonstrated that the loss of σB activity allowed mutants to take over a population of parental bacteria. Together, our results suggest that mutations affecting σB activity can arise during laboratory culture because of the growth advantage conferred by these mutations under mild stress conditions. The data highlight the significant cost of stress protection in this foodborne pathogen and emphasize the need for whole-genome sequence analysis of newly constructed strains to confirm the expected genotype.IMPORTANCE: In the present study, we investigated a collection of Listeria monocytogenes strains that all carried sigB operon mutations. The mutants all had reduced σB activity and were found to have a growth advantage under conditions of mild heat stress (42°C). In mixed cultures, these mutants outcompeted the wild type when mild heat stress was present but not at an optimal growth temperature. An analysis of 22,340 published L. monocytogenes genome sequences found a high rate of premature stop codons present in genes positively regulating σB activity. Together, these findings suggest that the occurrence of mutations that attenuate σB activity can be favored under conditions of mild stress, probably highlighting the burden on cellular resources that stems from deploying the general stress response.
|
|
| 7. |
- Hansen, Sabine, et al.
(författare)
-
A Novel Growth-Based Selection Strategy Identifies New Constitutively Active Variants of the Major Virulence Regulator PrfA in Listeria monocytogenes
- 2020
-
Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 202:11
-
Tidskriftsartikel (refereegranskat)abstract
- Listeria monocytogenes is a Gram-positive pathogen able to cause severe human infections. Its major virulence regulator is the transcriptional activator PrfA, a member of the Crp/Fnr family of transcriptional regulators. To establish a successful L. monocytogenes infection, the PrfA protein needs to be in an active conformation, either by binding the cognate inducer glutathione (GSH) or by possessing amino acid substitutions rendering the protein constitutively active (PrfA*). By a yet unknown mechanism, phosphotransferase system (PTS) sugars repress the activity of PrfA. We therefore took a transposon-based approach to identify the mechanism by which PTS sugars repress PrfA activity. For this, we screened a transposon mutant bank to identify clones able to grow in the presence of glucose-6-phosphate as the sole carbon source. Surprisingly, most of the isolated transposon mutants also carried amino acid substitutions in PrfA. In transposon-free strains, the PrfA amino acid substitution mutants displayed growth, virulence factor expression, infectivity, and DNA binding, agreeing with previously identified PrIA* mutants. Hence, the initial growth phenotype observed in the isolated clone was due to the amino acid substitution in PrfA and unrelated to the loci inactivated by the transposon mutant. Finally, we provide structural evidence for the existence of an intermediately activated PrfA state, which gives new insights into PrfA protein activation. IMPORTANCE The Gram-positive bacterium Listeria monocytogenes is a human pathogen affecting mainly the elderly, immunocompromised people, and pregnant women. It can lead to meningoencephalitis, septicemia, and abortion. The major virulence regulator in L. monocytogenes is the PrfA protein, a transcriptional activator. Using a growth-based selection strategy, we identified mutations in the PrfA protein leading to constitutively active virulence factor expression. We provide structural evidence for the existence of an intermediately activated PrfA state, which gives new insights into PrfA protein activation.
|
|
| 8. |
- Łebkowski, Tomasz, et al.
(författare)
-
AfsK-Mediated Site-Specific Phosphorylation Regulates DnaA Initiator Protein Activity in Streptomyces coelicolor
- 2020
-
Ingår i: Journal of Bacteriology. - 0021-9193. ; 202:3
-
Tidskriftsartikel (refereegranskat)abstract
- In all organisms, chromosome replication is regulated mainly at the initiation step. Most of the knowledge about the mechanisms that regulate replication initiation in bacteria has come from studies on rod-shaped bacteria, such as Escherichia coli and Bacillus subtilisStreptomyces is a bacterial genus that is characterized by distinctive features and a complex life cycle that shares some properties with the developmental cycle of filamentous fungi. The unusual lifestyle of streptomycetes suggests that these bacteria use various mechanisms to control key cellular processes. Here, we provide the first insights into the phosphorylation of the bacterial replication initiator protein, DnaA, from Streptomyces coelicolor We suggest that phosphorylation of DnaA triggers a conformational change that increases its ATPase activity and decreases its affinity for the replication origin, thereby blocking the formation of a functional orisome. We suggest that the phosphorylation of DnaA is catalyzed by Ser/Thr kinase AfsK, which was shown to regulate the polar growth of S. coelicolor Together, our results reveal that phosphorylation of the DnaA initiator protein functions as a negative regulatory mechanism to control the initiation of chromosome replication in a manner that presumably depends on the cellular localization of the protein.IMPORTANCE This work provides insights into the phosphorylation of the DnaA initiator protein in Streptomyces coelicolor and suggests a novel bacterial regulatory mechanism for initiation of chromosome replication. Although phosphorylation of DnaA has been reported earlier, its biological role was unknown. This work shows that upon phosphorylation, the cooperative binding of the replication origin by DnaA may be disturbed. We found that AfsK kinase is responsible for phosphorylation of DnaA. Upon upregulation of AfsK, chromosome replication occurred further from the hyphal tip. Orthologs of AfsK are exclusively found in mycelial actinomycetes that are related to Streptomyces and exhibit a complex life cycle. We propose that the AfsK-mediated regulatory pathway serves as a nonessential, energy-saving mechanism in S. coelicolor.
|
|
| 9. |
- Lima, Bruno P., et al.
(författare)
-
Streptococcus gordonii Poised for Glycan Feeding through a MUC5B-Discriminating, Lipoteichoic Acid-Mediated Outside-In Signaling Circuit
- 2022
-
Ingår i: Journal of Bacteriology. - : ASM International. - 0021-9193 .- 1098-5530. ; 204:6
-
Tidskriftsartikel (refereegranskat)abstract
- Many oral bacteria employ cell wall-anchored adhesins to bind to the salivary films coating the teeth and mucosal surfaces. Surface binding prevents clearance and facilitates catabolism of salivary film glycoproteins. We asked whether Streptococcus gordonii adhesin expression changes in response to surface salivary cues using a eukaryote-like, outside-in recognition and signaling circuit. To determine whether the cues were discriminated, S. gordonii was tested during cell adhesion and biofilm formation on a MUC5B-rich or lower-molecular-mass salivary fraction or an uncoated abiotic surface. Cells were recovered and analyzed for differences in gene expression and proteins in cell wall fractions. In salivary-free conditions, planktonic S. gordonii presented three prominent cell wall LPXTG-motif proteins, SGO_1487, SGO_0890, and MbpA (mucin-binding protein A; SGO_0707). During biofilm formation on MUC5B-coated surfaces, MbpA, a MUC5B-binding protein, and key genes in the tagatose and quorum-sensing pathways were strongly promoted. The response to MUC5B required the two-component system (TCS), streptococcal regulator of adhesins sensor and regulator (SraSR, SGO_1180/81), lipoteichoic acid (LTA), and the homologous paired adhesins, SspA and SspB (SspAB). LTA appears to link the outside signal (MUC5B) to intramembrane SraSR. Tagatose pathway gene expression may poise cells to metabolize MUC5B glycans and, with a quorum-sensing gene (luxS), may direct formation of a consortium to facilitate glycan cross-feeding by S. gordonii. We now show that a Gram-positive bacterium discriminates specific surface environmental cues using an outside-in signaling mechanism to apparently optimize colonization of saliva-coated surfaces. IMPORTANCE All organisms throughout the tree of life sense and respond to their surface environments. To discriminate among mucosal surface environmental cues, we report that Streptococcus gordonii recognizes a high-molecular-weight mucin glycoprotein, MUC5B, using the paired adhesins SspAB and lipoteichoic acid; the latter bridges the outside signal to an intramembrane two-component system to transcriptionally regulate a MUC5B-specific adhesin and genes that may facilitate glycan catabolism. All organisms throughout the tree of life sense and respond to their surface environments. To discriminate among mucosal surface environmental cues, we report that Streptococcus gordonii recognizes a high-molecular-weight mucin glycoprotein, MUC5B, using the paired adhesins SspAB and lipoteichoic acid; the latter bridges the outside signal to an intramembrane two-component system to transcriptionally regulate a MUC5B-specific adhesin and genes that may facilitate glycan catabolism.
|
|
| 10. |
- McGrath, Kaitlyn M., et al.
(författare)
-
Fitness benefits of a synonymous substitution in an ancient EF-Tu gene depend on the genetic background
- 2024
-
Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530.
-
Tidskriftsartikel (refereegranskat)abstract
- Synonymous mutations are changes to DNA sequence, which occur within translated genes but which do not affect the protein sequence. Although often referred to as silent mutations, evidence suggests that synonymous mutations can affect gene expression, mRNA stability, and even translation efficiency. A collection of both experimental and bioinformatic data has shown that synonymous mutations can impact cell phenotype, yet less is known about the molecular mechanisms and potential of beneficial or adaptive effects of such changes within evolved populations. Here, we report a beneficial synonymous mutation acquired via experimental evolution in an essential gene variant encoding the translation elongation factor protein EF-Tu. We demonstrate that this particular synonymous mutation increases EF-Tu mRNA and protein levels as well as global polysome abundance on RNA transcripts. Although presence of the synonymous mutation is clearly causative of such changes, we also demonstrate that fitness benefits are highly contingent on other potentiating mutations present within the genetic background in which the mutation arose. Our results underscore the importance of beneficial synonymous mutations, especially those that affect levels of proteins that are key for cellular processes.
|
|
