scholarly journals Linking the Salmonella enterica 1,2-propanediol utilization bacterial microcompartment shell to the enzymatic core via the shell protein PduB

2021 ◽  
Author(s):  
Nolan W Kennedy ◽  
Carolyn E Mills ◽  
Charlotte H Abrahamson ◽  
Andre Archer ◽  
Michael C Jewett ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Carbon Sources ◽  
Enzyme Concentration ◽  
Fluorescent Reporters ◽  
Shell Protein ◽  
Bacterial Microcompartment ◽  
Enzyme Encapsulation ◽  
Transmission Electron ◽  
Serovar Typhimurium ◽  
Enzymatic Machinery

Bacterial microcompartments (MCPs) are protein-based organelles that house the enzymatic machinery for metabolism of niche carbon sources, allowing enteric pathogens to outcompete native microbiota during host colonization. While much progress has been made toward understanding MCP biogenesis, questions still remain regarding the mechanism by which core MCP enzymes are enveloped within the MCP protein shell. Here we explore the hypothesis that the shell protein PduB is responsible for linking the shell of the 1,2-propanediol utilization (Pdu) MCP from Salmonella enterica serovar Typhimurium LT2 to its enzymatic core. Using fluorescent reporters, we demonstrate that all members of the Pdu enzymatic core are encapsulated in Pdu MCPs. We also demonstrate that PduB is the sole protein responsible for linking the entire Pdu enzyme core to the MCP shell. Using MCP purifications, transmission electron microscopy, and fluorescence microscopy we find that shell assembly can be decoupled from the enzymatic core, as apparently empty MCPs are formed in Salmonella strains lacking PduB. Mutagenesis studies also reveal that PduB is incorporated into the Pdu MCP shell via a conserved, lysine-mediated hydrogen bonding mechanism. Finally, growth assays and systems-level pathway modeling reveal that unencapsulated pathway performance is strongly impacted by enzyme concentration, highlighting the importance of minimizing polar effects when conducting these functional assays. Together, these results provide insight into the mechanism of enzyme encapsulation within Pdu MCPs and demonstrate that the process of enzyme encapsulation and shell assembly are separate processes in this system, a finding that will aid future efforts to understand MCP biogenesis.

scholarly journals Extracellular Polysaccharides Associated with Thin Aggregative Fimbriae of Salmonella enterica Serovar Enteritidis

2003 ◽  
Vol 185 (18) ◽  
pp. 5398-5407 ◽  
Author(s):  
A. P. White ◽  
D. L. Gibson ◽  
S. K. Collinson ◽  
P. A. Banser ◽  
W. W. Kay
Keyword(s):  
Salmonella Enterica ◽  
Extracellular Polysaccharide ◽  
Immune Serum ◽  
Extracellular Polysaccharides ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Western Blots ◽  
E Coli ◽  
Transmission Electron ◽  
Serovar Typhimurium ◽  
K 12

ABSTRACT Lipopolysaccharide (LPS) O polysaccharide was identified as the principle factor impeding intercellular formation of intact thin aggregative fimbriae (Tafi) in Salmonella enterica serovar Enteritidis. The extracellular nucleation-precipitation assembly pathway for these organelles was investigated by quantifying fimbrial formation between ΔagfA (AgfA recipient) and ΔagfB (AgfA donor) cells harboring mutations in LPS (galE::Tn10) and/or cellulose (ΔbcsA) synthesis. Intercellular complementation could be detected between ΔagfA and ΔagfB strains only when both possessed the galE mutation. LPS O polysaccharide appears to be an impenetrable barrier to AgfA assembly between cells but not within individual cells. The presence of cellulose did not restrict Tafi formation between cells. Transmission electron microscopy of w + S. enterica serovar Enteritidis 3b cells revealed diffuse Tafi networks without discernible fine structure. In the absence of cellulose, however, individual Tafi fibers were clearly visible, appeared to be occasionally branched, and showed the generally distinctive appearance described for Escherichia coli K-12 curli. A third extracellular matrix component closely associated with cellulose and Tafi was detected on Western blots by using immune serum raised to whole, purified Tafi aggregates. Cellulose was required to tightly link this material to cells. Antigenically similar material was also detected in S. enterica serovar Typhimurium and one diarrheagenic E. coli isolate. Preliminary analysis indicated that this material represented an anionic, extracellular polysaccharide that was distinct from colanic acid. Therefore, Tafi in their native state appear to exist as a complex with cellulose and at least one other component.

scholarly journals A Functional cra Gene Is Required forSalmonella enterica Serovar Typhimurium Virulence in BALB/c Mice

2000 ◽  
Vol 68 (6) ◽  
pp. 3772-3775 ◽  
Author(s):  
James H. Allen ◽  
Maryjane Utley ◽  
Han van den Bosch ◽  
Piet Nuijten ◽  
Maarten Witvliet ◽  
...  
Keyword(s):  
Carbon Metabolism ◽  
Salmonella Enterica ◽  
Protein A ◽  
Carbon Sources ◽  
Central Carbon Metabolism ◽  
Central Carbon ◽  
Serovar Typhimurium ◽  
Gluconeogenic Substrates

ABSTRACT A minitransposon mutant of Salmonella enterica serovar Typhimurium SR-11, SR-11 Fad−, is unable to utilize gluconeogenic substrates as carbon sources and is avirulent and immunogenic when administered perorally to BALB/c mice (M. J. Utley et al., FEMS Microbiol. Lett., 163:129–134, 1998). Here, evidence is presented that the mutation in SR-11 Fad− that renders the strain avirulent is in the cra gene, which encodes the Cra protein, a regulator of central carbon metabolism.

scholarly journals Role of Gluconeogenesis and the Tricarboxylic Acid Cycle in the Virulence of Salmonella enterica Serovar Typhimurium in BALB/c Mice

2006 ◽  
Vol 74 (2) ◽  
pp. 1130-1140 ◽  
Author(s):  
Merlin Tchawa Yimga ◽  
Mary P. Leatham ◽  
James H. Allen ◽  
David C. Laux ◽  
Tyrrell Conway ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Tricarboxylic Acid Cycle ◽  
Carbon Sources ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Glyoxylate Bypass ◽  
The Tca Cycle ◽  
Mouse Tissues ◽  
Serovar Typhimurium ◽  
Genes Encoding

ABSTRACT In Salmonella enterica serovar Typhimurium, the Cra protein (catabolite repressor/activator) regulates utilization of gluconeogenic carbon sources by activating transcription of genes in the gluconeogenic pathway, the glyoxylate bypass, the tricarboxylic acid (TCA) cycle, and electron transport and repressing genes encoding glycolytic enzymes. A serovar Typhimurium SR-11 Δcra mutant was recently reported to be avirulent in BALB/c mice via the peroral route, suggesting that gluconeogenesis may be required for virulence. In the present study, specific SR-11 genes in the gluconeogenic pathway were deleted (fbp, glpX, ppsA, and pckA), and the mutants were tested for virulence in BALB/c mice. The data show that SR-11 does not require gluconeogenesis to retain full virulence and suggest that as yet unidentified sugars are utilized by SR-11 for growth during infection of BALB/c mice. The data also suggest that the TCA cycle operates as a full cycle, i.e., a sucCD mutant, which prevents the conversion of succinyl coenzyme A to succinate, and an ΔsdhCDA mutant, which blocks the conversion of succinate to fumarate, were both attenuated, whereas both an SR-11 ΔaspA mutant and an SR-11 ΔfrdABC mutant, deficient in the ability to run the reductive branch of the TCA cycle, were fully virulent. Moreover, although it appears that SR-11 replenishes TCA cycle intermediates from substrates present in mouse tissues, fatty acid degradation and the glyoxylate bypass are not required, since an SR-11 ΔfadD mutant and an SR-11 ΔaceA mutant were both fully virulent.

scholarly journals Shifts from glucose to certain secondary carbon-sources result in activation of the extracytoplasmic function sigma factor σ E in Salmonella enterica serovar Typhimurium

Microbiology ◽  
2005 ◽  
Vol 151 (7) ◽  
pp. 2373-2383 ◽  
Author(s):  
William J. Kenyon ◽  
Sheena M. Thomas ◽  
Erin Johnson ◽  
Mark J. Pallen ◽  
Michael P. Spector
Keyword(s):  
Energy Source ◽  
Sigma Factor ◽  
Salmonella Enterica ◽  
Cell Envelope ◽  
Carbon Sources ◽  
Growth Conditions ◽  
Serovar Typhimurium ◽  
Lag Period ◽  
Diauxic Lag ◽  
Sustained Increase

Salmonella enterica serovar Typhimurium (S. Typhimurium) elicits the starvation-stress response (SSR) due to starvation for an essential nutrient, e.g. a carbon/energy source (C-source). As part of the SSR, the alternative sigma factor σ E is activated and induced. The authors suspect that this activation is, in part, triggered by changes in the S. Typhimurium cell envelope occurring during the adaptation from growth to carbon/energy starvation (C-starvation), and resulting in an increased need for σ E-regulated factors involved in the proper folding and assembly of newly synthesized proteins destined for this extracytoplasmic compartment. This led to the hypothesis that a σ E activation signal might arise during C-source shifts that cause the induction of proteins localized to the extracytoplasmic compartment, i.e. the outer membrane or periplasm, of the cell. To test this hypothesis, cultures were grown in minimal medium containing enough glucose to reach mid-exponential-phase, plus a non-limiting amount of a secondary ‘less-preferred’ but utilizable carbon/energy source. The σ E activity was then monitored using plasmids carrying rpoEP1– and rpoEP2–lacZ transcriptional fusions, which exhibit σ E-independent and -dependent lacZ expression, respectively. The secondary C-sources maltose, succinate and citrate, which have extracytoplasmic components involved in their utilization (e.g. LamB), resulted in a discernible diauxic lag period and a sustained increase in σ E activity. Growth transition from glucose to other utilizable phosphotransferase (PTS) and non-PTS C-sources, such as trehalose, mannose, mannitol, fructose, glycerol, d-galactose or l-arabinose, did not cause a discernible diauxic lag period or a sustained increase in σ E activity. Interestingly, a shift from glucose to melibiose, which does not use an extracytoplasmic-localized protein for uptake, did cause an observable diauxic lag period but did not result in a sustained increase in σ E activity. In addition, overexpression of LamB from an arabinose-inducible promoter leads to a significant increase in σ E activity in the absence of a glucose to maltose shift or C-starvation. Furthermore, a ΔlamB : : Ω-Kmr mutant, lacking the LamB maltoporin, exhibited an approximately twofold reduction in the sustained σ E activity observed during a glucose to maltose shift, again supporting the hypothesis. Interestingly, the LamB protein lacks the typical Y-X-F terminal tripeptide of the OmpC-like peptides that activate DegS protease activity leading to σ E activation. It does, however, possess a terminal pentapeptide (Q-M-E-I-W-W) that may function as a ligand for a putative class II PDZ-binding site. The authors therefore propose that the σ E regulon of S. Typhimurium not only is induced in response to deleterious environmental conditions, but also plays a role in the adaptation of cells to new growth conditions that necessitate changes in the extracytoplasmic compartment of the cell, which may involve alternative signal recognition and activation pathways that are independent of DegS.

scholarly journals Changes in Ultrastructure and Fourier Transform Infrared Spectrum of Salmonella enterica Serovar Typhimurium Cells after Exposure to Stress Conditions

2010 ◽  
Vol 76 (22) ◽  
pp. 7598-7607 ◽  
Author(s):  
A. Alvarez-Ordóñez ◽  
M. Prieto
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Fourier Transform ◽  
Stress Response ◽  
Salmonella Enterica ◽  
Fourier Transform Infrared ◽  
Ultrastructural Changes ◽  
Transmission Electron ◽  
Serovar Typhimurium ◽  
Ft Ir

ABSTRACT The effect of exposure to acid (pH 2.5), alkaline (pH 11.0), heat (55°C), and oxidative (40 mM H2O2) lethal conditions on the ultrastructure and global chemical composition of Salmonella enterica serovar Typhimurium CECT 443 cells was studied using transmission electron microscopy and Fourier transform infrared spectroscopy (FT-IR) combined with multivariate statistical methods (hierarchical cluster analysis and factor analysis). Infrared spectra exhibited marked differences in the five spectral regions for all conditions tested compared to those of nontreated control cells, which suggests the existence of a complex bacterial stress response in which modifications in a wide variety of cellular compounds are involved. The visible spectral changes observed in all of the spectral regions, together with ultrastructural changes observed by transmission electron microscopy and data obtained from membrane integrity tests, indicate the existence of membrane damage or alterations in membrane composition after heat, acid, alkaline, and oxidative treatments. Results obtained in this study indicate the potential of FT-IR spectroscopy to discriminate between intact and injured bacterial cells and between treatment technologies, and they show the adequacy of this technique to study the molecular aspects of bacterial stress response.

scholarly journals 1-Methylguanosine-Deficient tRNA of Salmonella enterica Serovar Typhimurium Affects Thiamine Metabolism

2003 ◽  
Vol 185 (3) ◽  
pp. 750-759 ◽  
Author(s):  
Glenn R. Björk ◽  
Kristina Nilsson
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Slow Growth ◽  
Carbon Sources ◽  
Gene Mutations ◽  
Gene Encoding ◽  
Translation Fidelity ◽  
Serovar Typhimurium ◽  
Purine Pathway ◽  
The Common

ABSTRACT In Salmonella enterica serovar Typhimurium a mutation in the purF gene encoding the first enzyme in the purine pathway blocks, besides the synthesis of purine, the synthesis of thiamine when glucose is used as the carbon source. On carbon sources other than glucose, a purF mutant does not require thiamine, since the alternative pyrimidine biosynthetic (APB) pathway is activated. This pathway feeds into the purine pathway just after the PurF biosynthetic step and upstream of the intermediate 4-aminoimidazolribotide, which is the common intermediate in purine and thiamine synthesis. The activity of this pathway is also influenced by externally added pantothenate. tRNAs from S. enterica specific for leucine, proline, and arginine contain 1-methylguanosine (m1G37) adjacent to and 3′ of the anticodon (position 37). The formation of m1G37 is catalyzed by the enzyme tRNA(m1G37)methyltransferase, which is encoded by the trmD gene. Mutations in this gene, which result in an m1G37 deficiency in the tRNA, in a purF mutant mediate PurF-independent thiamine synthesis. This phenotype is specifically dependent on the m1G37 deficiency, since several other mutations which also affect translation fidelity and induce slow growth did not cause PurF-independent thiamine synthesis. Some antibiotics that are known to reduce the efficiency of translation also induce PurF-independent thiamine synthesis. We suggest that a slow decoding event at a codon(s) read by a tRNA(s) normally containing m1G37 is responsible for the PurF-independent thiamine synthesis and that this event causes a changed flux in the APB pathway.

scholarly journals Glucose and glucose 6-phosphate as carbon sources in extra- and intracellular growth of enteroinvasive Escherichia coli and Salmonella enterica

Microbiology ◽  
2010 ◽  
Vol 156 (4) ◽  
pp. 1176-1187 ◽  
Author(s):  
Andreas Götz ◽  
Werner Goebel
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Carbon Sources ◽  
Enteric Bacteria ◽  
Wild Type ◽  
Intracellular Replication ◽  
Inhibition Of Growth ◽  
Serovar Typhimurium ◽  
Type Strains

To study the role of carbohydrates, in particular glucose, glucose 6-phosphate and mannose, as carbon substrates for extra- and intracellular replication of facultative intracellular enteric bacteria, mutants of two enteroinvasive Escherichia coli (EIEC) strains and a Salmonella enterica serovar Typhimurium isolate were constructed that were defective in the uptake of glucose and mannose (ΔptsG, manXYZ), glucose 6-phosphate (ΔuhpT) or all three carbohydrates (ΔptsG, manXYZ, uhpT). The ability of these mutants to grow in RPMI medium containing the respective carbohydrates and in Caco-2 cells was compared with that of the corresponding wild-type strains. In the three strains, deletions of ptsG, manXYZ or uhpT resulted in considerably different levels of inhibition of growth in vitro in the presence of glucose, mannose and glucose 6-phosphate, respectively, but hardly reduced their capability for intracellular replication in Caco-2 cells. Even the triple mutants ΔptsG, manXYZ, uhpT of the three enterobacterial strains were still able to replicate in Caco-2 cells, albeit at strain-specific lower rates than the corresponding wild-type strains.

scholarly journals pfs-Dependent Regulation of Autoinducer 2 Production in Salmonella enterica Serovar Typhimurium

2002 ◽  
Vol 184 (13) ◽  
pp. 3450-3456 ◽  
Author(s):  
Anne L. Beeston ◽  
Michael G. Surette
Keyword(s):  
Gene Product ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Regulation Of Gene Expression ◽  
Carbon Sources ◽  
Homoserine Lactone ◽  
Bacterial Cells ◽  
Autoinducer 2 ◽  
Serovar Typhimurium ◽  
Signal Synthesis

ABSTRACT Bacterial intercellular communication provides a mechanism for signal-dependent regulation of gene expression to promote coordinated population behavior. Salmonella enterica serovar Typhimurium produces a non-homoserine lactone autoinducer in exponential phase as detected by a Vibrio harveyi reporter assay for autoinducer 2 (AI-2) (M. G. Surette and B. L. Bassler, Proc. Natl. Acad. Sci. USA 95:7046-7050, 1998). The luxS gene product mediates the production of AI-2 (M. G. Surette, M. B. Miller, and B. L. Bassler, Proc. Natl. Acad. Sci. USA 96:1639-1644, 1999). Environmental cues such as rapid growth, the presence of preferred carbon sources, low pH, and/or high osmolarity were found to influence the production of AI-2 (M. G. Surette and B. L. Bassler, Mol. Microbiol. 31:585-595, 1999). In addition to LuxS, the pfs gene product (Pfs) is required for AI-2 production, as well as S-adenosylhomocysteine (SAH) (S. Schauder, K. Shokat, M. G. Surette, and B. L. Bassler, Mol. Microbiol. 41:463-476, 2001). In bacterial cells, Pfs exhibits both 5′-methylthioadenosine (MTA) and SAH nucleosidase functions. Pfs is involved in methionine metabolism, regulating intracellular MTA and SAH levels (elevated levels of MTA and SAH are potent inhibitors of polyamine synthetases and S-adenosylmethionine dependent methyltransferase reactions, respectively). To further investigate regulation of AI-2 production in Salmonella, we constructed pfs and luxS promoter fusions to a luxCDABE reporter in a low-copy-number vector, allowing an examination of transcription of the genes in the pathway for signal synthesis. Here we report that luxS expression is constitutive but that the transcription of pfs is tightly correlated to AI-2 production in Salmonella serovar Typhimurium 14028. Neither luxS nor pfs expression appears to be regulated by AI-2. These results suggest that AI-2 production is regulated at the level of LuxS substrate availability and not at the level of luxS expression. Our results indicate that AI-2-dependent signaling is a reflection of metabolic state of the cell and not cell density.

scholarly journals PduA Is a Shell Protein of Polyhedral Organelles Involved in Coenzyme B12-Dependent Degradation of 1,2-Propanediol in Salmonella enterica Serovar Typhimurium LT2

2002 ◽  
Vol 184 (5) ◽  
pp. 1253-1261 ◽  
Author(s):  
Gregory D. Havemann ◽  
Edith M. Sampson ◽  
Thomas A. Bobik
Keyword(s):  
Salmonella Enterica ◽  
Type Strain ◽  
Wild Type Strain ◽  
Polyclonal Antibodies ◽  
Wild Type ◽  
Shell Protein ◽  
Low Concentrations ◽  
Subtraction Procedure ◽  
Serovar Typhimurium ◽  
Coenzyme B

ABSTRACT Salmonella enterica forms polyhedral organelles involved in coenzyme B12-dependent 1,2-propanediol degradation. These organelles are thought to consist of a proteinaceous shell that encases coenzyme B12-dependent diol dehydratase and perhaps other enzymes involved in 1,2-propanediol degradation. The function of these organelles is unknown, and no detailed studies of their structure have been reported. Genes needed for organelle formation and for 1,2-propanediol degradation are located at the 1,2-propanediol utilization (pdu) locus, but the specific genes involved in organelle formation have not been identified. Here, we show that the pduA gene encodes a shell protein required for the formation of polyhedral organelles involved in coenzyme B12-dependent 1,2-propanediol degradation. A His6-PduA fusion protein was purified from a recombinant Escherichia coli strain and used for the preparation of polyclonal antibodies. The anti-PduA antibodies obtained were partially purified by a subtraction procedure and used to demonstrate that the PduA protein localized to the shell of the polyhedral organelles. In addition, electron microscopy studies established that strains with nonpolar pduA mutations were unable to form organelles. These results show that the pduA gene is essential for organelle formation and indicate that the PduA protein is a structural component of the shell of these organelles. Physiological studies of nonpolar pduA mutants were also conducted. Such mutants grew similarly to the wild-type strain at low concentrations of 1,2-propanediol but exhibited a period of interrupted growth in the presence of higher concentrations of this growth substrate. Growth tests also showed that a nonpolar pduA deletion mutant grew faster than the wild-type strain at low vitamin B12 concentrations. These results suggest that the polyhedral organelles formed by S. enterica during growth on 1,2-propanediol are not involved in the concentration of 1,2-propanediol or coenzyme B12, but are consistent with the hypothesis that these organelles moderate aldehyde production to minimize toxicity.

scholarly journals Salmonella enterica Serovar Typhimurium Colonizing the Lumen of the Chicken Intestine Grows Slowly and Upregulates a Unique Set of Virulence and Metabolism Genes

2011 ◽  
Vol 79 (10) ◽  
pp. 4105-4121 ◽  
Author(s):  
P. C. Harvey ◽  
M. Watson ◽  
S. Hulme ◽  
M. A. Jones ◽  
M. Lovell ◽  
...  
Keyword(s):  
Cell Division ◽  
Salmonella Enterica ◽  
Carbon Sources ◽  
Global Gene Expression ◽  
Intestinal Lumen ◽  
Broth Culture ◽  
Content Type ◽  
Serovar Typhimurium

ABSTRACTThe pattern of global gene expression inSalmonella entericaserovar Typhimurium bacteria harvested from the chicken intestinal lumen (cecum) was compared with that of a late-log-phase LB broth culture using a whole-genome microarray. Levels of transcription, translation, and cell divisionin vivowere lower than thosein vitro.S.Typhimurium appeared to be using carbon sources, such as propionate, 1,2-propanediol, and ethanolamine, in addition to melibiose and ascorbate, the latter possibly transformed tod-xylulose. Amino acid starvation appeared to be a factor during colonization. Bacteria in the lumen were non- or weakly motile and nonchemotactic but showed upregulation of a number of fimbrial andSalmonellapathogenicity island 3 (SPI-3) and 5 genes, suggesting a close physical association with the host during colonization.S.Typhimurium bacteria harvested from the cecal mucosa showed an expression profile similar to that of bacteria from the intestinal lumen, except that levels of transcription, translation, and cell division were higher and glucose may also have been used as a carbon source.

Sign in / Sign up

Export Citation Format

Share Document