High-Salinity Growth Conditions Promote Tat-Independent Secretion of Tat Substrates in Bacillus subtilis

ABSTRACTThe Gram-positive bacteriumBacillus subtiliscontains two Tat translocases, which can facilitate transport of folded proteins across the plasma membrane. Previous research has shown that Tat-dependent protein secretion inB. subtilisis a highly selective process and that heterologous proteins, such as the green fluorescent protein (GFP), are poor Tat substrates in this organism. Nevertheless, when expressed inEscherichia coli, bothB. subtilisTat translocases facilitated exclusively Tat-dependent export of folded GFP when the twin-arginine (RR) signal peptides of theE. coliAmiA, DmsA, or MdoD proteins were attached. Therefore, the present studies were aimed at determining whether the same RR signal peptide-GFP precursors would also be exported Tat dependently inB. subtilis. In addition, we investigated the secretion of GFP fused to the full-length YwbN protein, a strict Tat substrate inB. subtilis. Several investigated GFP fusion proteins were indeed secreted inB. subtilis, but this secretion was shown to be completely Tat independent. At high-salinity growth conditions, the Tat-independent secretion of GFP as directed by the RR signal peptides from theE. coliAmiA, DmsA, or MdoD proteins was significantly enhanced, and this effect was strongest in strains lacking the TatAy-TatCy translocase. This implies that high environmental salinity has a negative influence on the avoidance of Tat-independent secretion of AmiA-GFP, DmsA-GFP, and MdoD-GFP. We conclude that as-yet-unidentified control mechanisms reject the investigated GFP fusion proteins for translocation by theB. subtilisTat machinery and, at the same time, set limits to their Tat-independent secretion, presumably via the Sec pathway.

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Engineering of The Bacillus Subtilis PhoD Signal Peptide To Direct High-Level, Tat-Specific Export of A Single-Chain Antibody Fragment To The Periplasm in Eschericha Coli

10.21203/rs.3.rs-48566/v1 ◽

2020 ◽

Author(s):

Chillel Jawara ◽

Kirsty L Richards ◽

Amber R Peswani ◽

Kelly L Walker ◽

Lara Nascimento ◽

...

Keyword(s):

Bacillus Subtilis ◽

Signal Peptide ◽

Antibody Fragment ◽

Signal Peptides ◽

Heterologous Proteins ◽

Single Chain ◽

Single Chain Antibody ◽

E Coli ◽

Tat Pathway ◽

Single Chain Antibody Fragment

Abstract Background: Numerous high-value proteins have been produced in E. coli, and a favoured strategy is to export the protein of interest to the periplasm by means of an N-terminal signal peptide. While the Sec pathway has been extensively used for this purpose, the Tat pathway has potential because it transports fully-folded heterologous proteins. Most studies on the Tat pathway have used the E. coli TorA signal peptide to direct export, because it is highly Tat-specific, unlike many Tat signal peptides which can also function as Sec signal peptides. However, the TorA signal peptide is prone to degradation in the cytoplasm, leading to reduced export rates in some cases. Here, we have tested a range of alternative signal peptides for their ability to direct Tat-dependent export of a single-chain antibody fragment (scFv). Results: We show that the signal peptides of E. coli AmiC, MdoD and YcbK direct efficient export of the scFv by both the Tat and Sec pathways, which may be a disadvantage when Tat-specific export is required. The same applies to the Tat signal peptide of Bacillus subtilis PhoD, which likewise directs efficient export by Sec. We engineered the PhoD signal peptide by introduction of a Lys or Asn residue in the C-terminal domain of the signal peptide, and we show that this substitution renders the signal peptide Tat-specific. These signal peptides, designated PhoDk and PhoDn, direct efficient export of scFv in shake flask and fed-batch fermentation studies, reaching export levels that are well above those obtained with the TorA signal peptide. Culturing in ambr250 bioreactors was used to fine-tune the growth conditions, and the net result was export of the scFv by the Tat pathway at levels of approximately 1g protein/L culture. Conclusions: The new PhoDn and PhoDk signal peptides have significant potential for the export of heterologous proteins by the Tat system.

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Functional Implementation of the Posttranslational SecB-SecA Protein-Targeting Pathway in Bacillus subtilis

Applied and Environmental Microbiology ◽

10.1128/aem.07209-11 ◽

2011 ◽

Vol 78 (3) ◽

pp. 651-659 ◽

Cited By ~ 18

Author(s):

Liuyang Diao ◽

Qilei Dong ◽

Zhaohui Xu ◽

Sheng Yang ◽

Jiahai Zhou ◽

...

Keyword(s):

Bacillus Subtilis ◽

Protein Targeting ◽

Heterologous Proteins ◽

Gram Positive ◽

Content Type ◽

E Coli ◽

New Strategy ◽

Secretory Production ◽

Close Relatives

ABSTRACTBacillus subtilisand its close relatives are widely used in industry for the Sec-dependent secretory production of proteins. Like other Gram-positive bacteria,B. subtilisdoes not possess SecB, a dedicated targeting chaperone that posttranslationally delivers exported proteins to the SecA component of the translocase. In the present study, we have implemented a functional SecB-dependent protein-targeting pathway intoB. subtilisby coexpressing SecB fromEscherichia colitogether with a SecA hybrid protein in which the carboxyl-terminal 32 amino acids of theB. subtilisSecA were replaced by the corresponding part of SecA fromE. coli.In vitropulldown experiments showed that, in contrast toB. subtilisSecA, the hybrid SecA protein gained the ability to efficiently bind toE. coliSecB, suggesting that the structural details of the extreme C-terminal region of SecA constitute a crucial SecB binding specificity determinant. Using a poorly exported mutant maltose binding protein (MalE11) and alkaline phosphatase (PhoA) as model proteins, we could demonstrate that the secretion of both proteins byB. subtiliswas significantly enhanced in the presence of the artificial protein targeting pathway. Mutations in SecB that do not influence its chaperone activity but prevent its interaction with SecA abolished the secretion stimulation of both proteins, demonstrating that the implemented pathway in fact critically depends on the SecB targeting function. From a biotechnological view, our results open up a new strategy for the improvement of Gram-positive bacterial host systems for the secretory production of heterologous proteins.

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Engineering of the Bacillus subtilis PhoD signal peptide to direct high-level, Tat-specific export of a single-chain antibody fragment to the periplasm in Eschericha coli

10.21203/rs.3.rs-48566/v2 ◽

2020 ◽

Author(s):

Chillel Jawara ◽

Kirsty L Richards ◽

Amber R Peswani ◽

Kelly L Walker ◽

Lara Nascimento ◽

...

Keyword(s):

Bacillus Subtilis ◽

Signal Peptide ◽

Antibody Fragment ◽

Signal Peptides ◽

Heterologous Proteins ◽

Single Chain ◽

Single Chain Antibody ◽

E Coli ◽

Tat Pathway ◽

Single Chain Antibody Fragment

Abstract Background : Numerous high-value proteins have been produced in E. coli, and a favoured strategy is to export the protein of interest to the periplasm by means of an N-terminal signal peptide. While the Sec pathway has been extensively used for this purpose, the Tat pathway has potential because it transports fully-folded heterologous proteins. Most studies on the Tat pathway have used the E. coli TorA signal peptide to direct export, because it is highly Tat-specific, unlike many Tat signal peptides which can also function as Sec signal peptides. However, the TorA signal peptide is prone to degradation in the cytoplasm, leading to reduced export rates in some cases. Here, we have tested a range of alternative signal peptides for their ability to direct Tat-dependent export of a single-chain antibody fragment (scFv). Results : We show that the signal peptides of E. coli AmiC, MdoD and YcbK direct efficient export of the scFv by both the Tat and Sec pathways, which may be a disadvantage when Tat-specific export is required. The same applies to the Tat signal peptide of Bacillus subtilis PhoD, which likewise directs efficient export by Sec. We engineered the PhoD signal peptide by introduction of a Lys or Asn residue in the C-terminal domain of the signal peptide, and we show that this substitution renders the signal peptide Tat-specific. These signal peptides, designated PhoDk and PhoDn, direct efficient export of scFv in shake flask and fed-batch fermentation studies, reaching export levels that are well above those obtained with the TorA signal peptide. Culturing in ambr250 bioreactors was used to fine-tune the growth conditions, and the net result was export of the scFv by the Tat pathway at levels of approximately 1g protein/L culture. Conclusions : The new PhoDn and PhoDk signal peptides have significant potential for the export of heterologous proteins by the Tat system.

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Synthesis, Release, and Recapture of Compatible Solute Proline by Osmotically Stressed Bacillus subtilis Cells

Applied and Environmental Microbiology ◽

10.1128/aem.01040-12 ◽

2012 ◽

Vol 78 (16) ◽

pp. 5753-5762 ◽

Cited By ~ 41

Author(s):

Tamara Hoffmann ◽

Carsten von Blohn ◽

Agnieszka Stanek ◽

Susanne Moses ◽

Helena Barzantny ◽

...

Keyword(s):

Bacillus Subtilis ◽

High Salinity ◽

De Novo ◽

Compatible Solutes ◽

Growth Conditions ◽

Compatible Solute ◽

Content Type ◽

Cellular Defense ◽

Stressed Cells ◽

External Salinity

ABSTRACTBacillus subtilissynthesizes large amounts of the compatible solute proline as a cellular defense against high osmolarity to ensure a physiologically appropriate level of hydration of the cytoplasm and turgor. It also imports proline for this purpose via the osmotically inducible OpuE transport system. Unexpectedly, anopuEmutant was at a strong growth disadvantage in high-salinity minimal media lacking proline. Appreciable amounts of proline were detected in the culture supernatant of theopuEmutant strain, and they rose concomitantly with increases in the external salinity. We found that the intracellular proline pool of severely salinity-stressed cells of theopuEmutant was considerably lower than that of itsopuE+parent strain. This loss of proline into the medium and the resulting decrease in the intracellular proline content provide a rational explanation for the observed salt-sensitive growth phenotype of cells lacking OpuE. None of the known MscL- and MscS-type mechanosensitive channels ofB. subtilisparticipated in the release of proline under permanently imposed high-salinity growth conditions. The data reported here show that the OpuE transporter not only possesses the previously reported role for the scavenging of exogenously provided proline as an osmoprotectant but also functions as a physiologically highly important recapturing device for proline that is synthesizedde novoand subsequently released by salt-stressedB. subtiliscells. The wider implications of our findings for the retention of compatible solutes by osmotically challenged microorganisms and the roles of uptake systems for compatible solutes are considered.

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A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽

10.1128/mbio.01105-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 7

Author(s):

Christopher W. Lennon ◽

Kimberly C. Lemmer ◽

Jessica L. Irons ◽

Max I. Sellman ◽

Timothy J. Donohue ◽

...

Keyword(s):

Escherichia Coli ◽

Structure Function ◽

Rhodobacter Sphaeroides ◽

Fatty Acid Content ◽

Regulatory Protein ◽

Growth Conditions ◽

Globular Domain ◽

Content Type ◽

E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

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Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01549-10 ◽

2011 ◽

Vol 55 (5) ◽

pp. 2438-2441 ◽

Cited By ~ 107

Author(s):

Zeynep Baharoglu ◽

Didier Mazel

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Vibrio Cholerae ◽

Fluorescent Protein ◽

Resistance Development ◽

Content Type ◽

E Coli ◽

Wide Range ◽

Green Fluorescent ◽

Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

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Artificial Septal Targeting of Bacillus subtilis Cell Division Proteins in Escherichia coli: an Interspecies Approach to the Study of Protein-Protein Interactions in Multiprotein Complexes

Journal of Bacteriology ◽

10.1128/jb.00462-08 ◽

2008 ◽

Vol 190 (18) ◽

pp. 6048-6059 ◽

Cited By ~ 17

Author(s):

Carine Robichon ◽

Glenn F. King ◽

Nathan W. Goehring ◽

Jon Beckwith

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Cell Division ◽

Protein Interactions ◽

Fluorescent Protein ◽

Bacterial Cell Division ◽

Protein Protein Interactions ◽

Positive Interaction ◽

E Coli ◽

Multiprotein Complexes

ABSTRACT Bacterial cell division is mediated by a set of proteins that assemble to form a large multiprotein complex called the divisome. Recent studies in Bacillus subtilis and Escherichia coli indicate that cell division proteins are involved in multiple cooperative binding interactions, thus presenting a technical challenge to the analysis of these interactions. We report here the use of an E. coli artificial septal targeting system for examining the interactions between the B. subtilis cell division proteins DivIB, FtsL, DivIC, and PBP 2B. This technique involves the fusion of one of the proteins (the “bait”) to ZapA, an E. coli protein targeted to mid-cell, and the fusion of a second potentially interacting partner (the “prey”) to green fluorescent protein (GFP). A positive interaction between two test proteins in E. coli leads to septal localization of the GFP fusion construct, which can be detected by fluorescence microscopy. Using this system, we present evidence for two sets of strong protein-protein interactions between B. subtilis divisomal proteins in E. coli, namely, DivIC with FtsL and DivIB with PBP 2B, that are independent of other B. subtilis cell division proteins and that do not disturb the cytokinesis process in the host cell. Our studies based on the coexpression of three or four of these B. subtilis cell division proteins suggest that interactions among these four proteins are not strong enough to allow the formation of a stable four-protein complex in E. coli in contrast to previous suggestions. Finally, our results demonstrate that E. coli artificial septal targeting is an efficient and alternative approach for detecting and characterizing stable protein-protein interactions within multiprotein complexes from other microorganisms. A salient feature of our approach is that it probably only detects the strongest interactions, thus giving an indication of whether some interactions suggested by other techniques may either be considerably weaker or due to false positives.

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Droplet- Rather than Aerosol-Mediated Dispersion Is the Primary Mechanism of Bacterial Transmission from Contaminated Hand-Washing Sink Traps

Applied and Environmental Microbiology ◽

10.1128/aem.01997-18 ◽

2018 ◽

Vol 85 (2) ◽

Cited By ~ 9

Author(s):

Shireen M. Kotay ◽

Rodney M. Donlan ◽

Christine Ganim ◽

Katie Barry ◽

Bryan E. Christensen ◽

...

Keyword(s):

Patient Care ◽

Sampling Methods ◽

Fluorescent Protein ◽

Model Organism ◽

Air Sampling ◽

Multidrug Resistant ◽

Hand Washing ◽

Content Type ◽

E Coli ◽

Green Fluorescent

ABSTRACT An alarming rise in hospital outbreaks implicating hand-washing sinks has led to widespread acknowledgment that sinks are a major reservoir of antibiotic-resistant pathogens in patient care areas. An earlier study using green fluorescent protein (GFP)-expressing Escherichia coli (GFP-E. coli) as a model organism demonstrated dispersal from drain biofilms in contaminated sinks. The present study further characterizes the dispersal of microorganisms from contaminated sinks. Replicate hand-washing sinks were inoculated with GFP-E. coli, and dispersion was measured using qualitative (settle plates) and quantitative (air sampling) methods. Dispersal caused by faucet water was captured with settle plates and air sampling methods when bacteria were present on the drain. In contrast, no dispersal was captured without or in between faucet events, amending an earlier theory that bacteria aerosolize from the P-trap and disperse. Numbers of dispersed GFP-E. coli cells diminished substantially within 30 minutes after faucet usage, suggesting that the organisms were associated with larger droplet-sized particles that are not suspended in the air for long periods. IMPORTANCE Among the possible environmental reservoirs in a patient care environment, sink drains are increasingly recognized as a potential reservoir to hospitalized patients of multidrug-resistant health care-associated pathogens. With increasing antimicrobial resistance limiting therapeutic options for patients, a better understanding of how pathogens disseminate from sink drains is urgently needed. Once this knowledge gap has decreased, interventions can be engineered to decrease or eliminate transmission from hospital sink drains to patients. The current study further defines the mechanisms of transmission for bacteria that colonize sink drains.

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Organization of the Flagellar Switch Complex ofBacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.00626-18 ◽

2018 ◽

Vol 201 (8) ◽

Cited By ~ 13

Author(s):

Elizabeth Ward ◽

Eun A Kim ◽

Joseph Panushka ◽

Tayson Botelho ◽

Trevor Meyer ◽

...

Keyword(s):

Bacillus Subtilis ◽

Protein Interaction ◽

Protein Complex ◽

Cross Linking ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Middle Domain ◽

Flagellar Rotation

ABSTRACTWhile the protein complex responsible for controlling the direction (clockwise [CW] or counterclockwise [CCW]) of flagellar rotation has been fairly well studied inEscherichia coliandSalmonella, less is known about the switch complex inBacillus subtilisor other Gram-positive species. Two component proteins (FliG and FliM) are shared betweenE. coliandB. subtilis, but in place of the protein FliN found inE. coli, theB. subtiliscomplex contains the larger protein FliY. Notably, inB. subtilisthe signaling protein CheY-phosphate induces a switch from CW to CCW rotation, opposite to its action inE. coli. Here, we have examined the architecture and function of the switch complex inB. subtilisusing targeted cross-linking, bacterial two-hybrid protein interaction experiments, and characterization of mutant phenotypes. In major respects, theB. subtilisswitch complex appears to be organized similarly to that inE. coli. The complex is organized around a ring built from the large middle domain of FliM; this ring supports an array of FliG subunits organized in a similar way to that ofE. coli, with the FliG C-terminal domain functioning in the generation of torque via conserved charged residues. Key differences fromE. coliinvolve the middle domain of FliY, which forms an additional, more outboard array, and the C-terminal domains of FliM and FliY, which are organized into both FliY homodimers and FliM heterodimers. Together, the results suggest that the CW and CCW conformational states are similar in the Gram-negative and Gram-positive switches but that CheY-phosphate drives oppositely directed movements in the two cases.IMPORTANCEFlagellar motility plays key roles in the survival of many bacteria and in the harmful action of many pathogens. Bacterial flagella rotate; the direction of flagellar rotation is controlled by a multisubunit protein complex termed the switch complex. This complex has been extensively studied in Gram-negative model species, but little is known about the complex inBacillus subtilisor other Gram-positive species. Notably, the switch complex in Gram-positive species responds to its effector CheY-phosphate (CheY-P) by switching to CCW rotation, whereas inE. coliorSalmonellaCheY-P acts in the opposite way, promoting CW rotation. In the work here, the architecture of theB. subtilisswitch complex has been probed using cross-linking, protein interaction measurements, and mutational approaches. The results cast light on the organization of the complex and provide a framework for understanding the mechanism of flagellar direction control inB. subtilisand other Gram-positive species.

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Identification and Characterization of Genes Required for 5-Hydroxyuridine Synthesis in Bacillus subtilis and Escherichia coli tRNA

Journal of Bacteriology ◽

10.1128/jb.00433-19 ◽

2019 ◽

Vol 201 (20) ◽

Cited By ~ 7

Author(s):

Charles T. Lauhon

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Genomic Context ◽

Similar Reduction ◽

Content Type ◽

E Coli ◽

Wobble Position ◽

Fertile Ground ◽

And Function

ABSTRACT In bacteria, tRNAs that decode 4-fold degenerate family codons and have uridine at position 34 of the anticodon are typically modified with either 5-methoxyuridine (mo5U) or 5-methoxycarbonylmethoxyuridine (mcmo5U). These modifications are critical for extended recognition of some codons at the wobble position. Whereas the alkylation steps of these modifications have been described, genes required for the hydroxylation of U34 to give 5-hydroxyuridine (ho5U) remain unknown. Here, a number of genes in Escherichia coli and Bacillus subtilis are identified that are required for wild-type (wt) levels of ho5U. The yrrMNO operon is identified in B. subtilis as important for the biosynthesis of ho5U. Both yrrN and yrrO are homologs to peptidase U32 family genes, which includes the rlhA gene required for ho5C synthesis in E. coli. Deletion of either yrrN or yrrO, or both, gives a 50% reduction in mo5U tRNA levels. In E. coli, yegQ was found to be the only one of four peptidase U32 genes involved in ho5U synthesis. Interestingly, this mutant shows the same 50% reduction in (m)cmo5U as that observed for mo5U in the B. subtilis mutants. By analyzing the genomic context of yegQ homologs, the ferredoxin YfhL is shown to be required for ho5U synthesis in E. coli to the same extent as yegQ. Additional genes required for Fe-S biosynthesis and biosynthesis of prephenate give the same 50% reduction in modification. Together, these data suggest that ho5U biosynthesis in bacteria is similar to that of ho5C, but additional genes and substrates are required for complete modification. IMPORTANCE Modified nucleotides in tRNA serve to optimize both its structure and function for accurate translation of the genetic code. The biosynthesis of these modifications has been fertile ground for uncovering unique biochemistry and metabolism in cells. In this work, genes that are required for a novel anaerobic hydroxylation of uridine at the wobble position of some tRNAs are identified in both Bacillus subtilis and Escherichia coli. These genes code for Fe-S cluster proteins, and their deletion reduces the levels of the hydroxyuridine by 50% in both organisms. Additional genes required for Fe-S cluster and prephenate biosynthesis and a previously described ferredoxin gene all display a similar reduction in hydroxyuridine levels, suggesting that still other genes are required for the modification.

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