Instability of<i>ackA</i>(Acetate Kinase) Mutations and Their Effects on Acetyl Phosphate and ATP Amounts in<i>Streptococcus pneumoniae</i>D39

Ramos‐Montañez, Smirla; Kazmierczak, Krystyna M.; Hentchel, Kristy L.; Winkler, Malcolm E.

doi:10.1128/jb.00995-10

Cited by 49 publications

(90 citation statements)

References 53 publications

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“…The strains MK127 and MK128, containing a deletion mutation in the capsule locus, were made by transforming the strains JWV500 and MK119, respectively, with a PCR product causing replacement of the gene cps2E (encoding a glucose phosphotransferase which initiates capsule synthesis) with a kanamycin resistance gene (M. G. Jørgensen and J.-W. Veening, unpublished data). This deletion is similar to a deletion that was described previously by RamosMontañez et al (29).…”

Section: Methodssupporting

confidence: 60%

“…To do so, we introduced a capsule mutation in both the HlpA-GFP and HlpA-RFP reporter strains. The capsule mutation introduced was ⌬cps2E (here designated ⌬cps) which has been used in other studies (29,47) and has been shown to produce capsule-deficient cells. Next, we performed competition of bacterial adherence between HlpA-GFP (strain JWV500) and a ⌬cps strain expressing HlpA-RFP (⌬cps/ HlpA-RFP; MK128) or between HlpA-RFP (MK119) and a ⌬cps/ HlpA-GFP (MK127) strain and added them to a confluent A549 monolayer (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Bright Fluorescent Streptococcus pneumoniae for Live-Cell Imaging of Host-Pathogen Interactions

et al. 2015

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c Streptococcus pneumoniae is a common nasopharyngeal resident in healthy people but, at the same time, one of the major causes of infectious diseases such as pneumonia, meningitis, and sepsis. The shift from commensal to pathogen and its interaction with host cells are poorly understood. One of the major limitations for research on pneumococcal-host interactions is the lack of suitable tools for live-cell imaging. To address this issue, we developed a generally applicable strategy to create genetically stable, highly fluorescent bacteria. Our strategy relies on fusing superfolder green fluorescent protein (GFP) or a far-red fluorescent protein (RFP) to the abundant histone-like protein HlpA. Due to efficient translation and limited cellular diffusion of these fusions, the cells are 25-fold brighter than those of the currently best available imaging S. pneumoniae strain. These novel bright pneumococcal strains are fully virulent, and the GFP reporter can be used for in situ imaging in mouse tissue. We used our reporter strains to study the effect of the polysaccharide capsule, a major pneumococcal virulence factor, on different stages of infection. By dual-color live-cell imaging experiments, we show that unencapsulated pneumococci adhere significantly better to human lung epithelial cells than encapsulated strains, in line with previous data obtained by classical approaches. We also confirm with live-cell imaging that the capsule protects pneumococci from neutrophil phagocytosis, demonstrating the versatility and usability of our reporters. The described imaging tools will pave the way for live-cell imaging of pneumococcal infection and help further understanding of the mechanisms of pneumococcal pathogenesis. Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, and pneumococcal infections (e.g., pneumonia, septicemia, and meningitis) kill more than 1 million people every year (1). Pneumococci are also quiescent colonizers of the upper respiratory tract, particularly in children, but little is known about the mechanisms underlying the transition from commensal to pathogen. It is therefore of crucial importance to understand the entire pneumococcal pathogenesis cycle in detail.The polysaccharide capsule covering the cell surface is the most central virulence factor of S. pneumoniae. The involvement of the capsule in pneumococcal pathogenesis has been appreciated since Griffith in 1928 (2) published his famous transformation experiment with rough and smooth strains of S. pneumoniae. Today, it is known that the bulky capsule, which is either negatively charged or neutral, contributes to pathogenesis by protecting pneumococci against the human immune system. For example, the capsule hinders phagocytosis and inhibits complement activity (3-6). Over 90 different pneumococcal serotypes have been identified to date (7), and the different serotypes vary in how well they protect the bacteria against phagocytosis (6). Furthermore, the amount of capsule differs between bacteria, and it has been s...

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Section: Methodssupporting

confidence: 60%

Section: Resultsmentioning

confidence: 99%

Bright Fluorescent Streptococcus pneumoniae for Live-Cell Imaging of Host-Pathogen Interactions

et al. 2015

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“…This observation directly links the acetylphosphate level in the cytosol to dormancy control. Thus, intracellular accumulation of acetyl-phosphate might disturb global signal transduction and has a toxic effect on the cells (62,63), whereas lack of acetyl-phosphate might result in reduced phosphorylation of response regulators and thereby in altered gene expression. The inability of the ⌬pta knockout strain to grow in the presence of acetate might therefore result from an intracellular increase in the acetyl-phosphate.…”

Section: Discussionmentioning

confidence: 99%

Acetate Dissimilation and Assimilation in Mycobacterium tuberculosis Depend on Carbon Availability

et al. 2015

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Mycobacterium tuberculosis CoA) as a substrate, acetyl-phosphate is generated and finally dephosphorylated to acetate, which is secreted into the medium. Knockout mutants lacking either the pta or ackA gene showed significantly reduced acetate production when grown on fatty acids. This effect is even more pronounced when the glyoxylate shunt is blocked, resulting in higher acetate levels released to the medium. The secretion of acetate was followed by an assimilation of the metabolite when other carbon substrates became limiting. Our data indicate that during acetate assimilation, the Pta-AckA pathway acts in concert with another enzymatic reaction, namely, the acetyl-CoA synthetase (Acs) reaction. Thus, acetate metabolism might possess a dual function, mediating an overflow reaction to release excess carbon units and resumption of acetate as a carbon substrate. IMPORTANCEDuring infection, host-derived lipid components present the major carbon source at the infection site. ␤-Oxidation of fatty acids results in the formation of acetyl-CoA. In this study, we demonstrate that consumption of fatty acids by Mycobacterium tuberculosis activates an overflow mechanism, causing the pathogen to release excess carbon intermediates as acetate. The Pta-AckA pathway mediating acetate formation proved to be reversible, enabling M. tuberculosis to reutilize the previously secreted acetate as a carbon substrate for metabolism.

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“…Strains containing antibiotic markers were constructed by transforming linear DNA amplicons synthesized by overlapping fusion PCR into competent pneumococcal cells as described previously (43,49,51,53). Primers synthesized for this study are listed in Table S2 in the supplemental material.…”

Section: Methodsmentioning

confidence: 99%

“…The same patterns of transformation were observed in encapsulated strain D39 and derivatives, indicating that capsule did not play a role in the low rate of recovery of ⌬mreC, ⌬mreD, and ⌬mreCD mutants (see below and data not shown). The appearance of relatively low numbers of colonies of variable sizes after longer incubation is a hallmark of suppressor mutation accumulation (1,49).…”

Section: Vol 193 2011 Properties and Localization Of Pneumococcal Mmentioning

confidence: 99%

The Requirement for Pneumococcal MreC and MreD Is Relieved by Inactivation of the Gene Encoding PBP1a

2011

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Three basic patterns of peptidoglycan (PG) synthesis have emerged from the study of rod-shaped (bacillus), spherical (coccus), and ellipsoid (ovococcus) bacteria (reviewed in references 12, 71, and 72). In rod-shaped bacteria, like Escherichia coli, Bacillus subtilis, and Caulobacter crescentus, PG synthesis occurs at two locations catalyzed by distinct sets of proteins (12,35,36,71). Lateral PG synthesis elongates the side walls of these bacteria and results in their rod shape (12,35,71). Lateral PG synthesis is mediated by actin-like MreB paralogue proteins, which form filamentous helical structures in spirals over the length of cells (12,35,66). MreB is thought to organize the MreC and MreD proteins, which in turn recruit specific penicillin-binding proteins (PBPs) and other division proteins that catalyze lateral PG formation (26,54,55,70). Septal PG synthesis is organized by FtsZ ring formation, followed by the orderly assembly of the divisome complex, which includes a set of PBPs different from those involved in lateral PG synthesis (16,22,23). The mutational interruption of lateral or septal PG synthesis of rod-shaped bacteria generally results in the formation of spherical or elongated cells, respectively (3, 4, 9, 31). Although useful, this two-state model is obviously an oversimplification (35), and recent results reveal that MreB, MreC, MreD, and specific PBPs also form annular rings adjacent to FtsZ rings at the septa of dividing E. coli cells (67). Moreover, some PBPs and division regulators, such as PBP1 and GpsB of B. subtilis, shuttle between the lateral and septal PG synthesis machineries (9).Much less is known about the mechanisms of PG synthesis in coccus and ovococcus species. In spherical species, such as Staphylococcus aureus, only a septal PG synthesis machinery seems to be present (20,47,72), although S. aureus still encodes homologues of proteins like MreC and MreD that mediate lateral PG synthesis in rod-shaped bacteria (35). In contrast, ovococcus species, such as Streptococcus pneumoniae and Lactococcus lactis, form American football-shaped cells by a combination of peripheral sidewall and septal PG synthesis that occurs in the midcell regions of dividing cells (Fig. 1) (11,42). Ovococcus species lack an MreB homologue, and peripheral and septal PG synthesis are likely coordinated with and organized by FtsZ ring formation (64,65,72). Because two modes of PG biosynthesis are required to form ellipsoidshaped bacteria, models have been proposed for two different PG synthesis machineries (18,21,33,72). Based on the composition of the complexes in rod-shaped cells, it has been hypothesized that specific sets of homologous cell division proteins mediate peripheral (MreC, MreD, RodA, and PBP2b) and septal (FtsZ, EzrA, PBP2x, FtsW, and DivIB/FtsL/DivIC) PG synthesis in ovococcus bacteria (72). A recent study correlating the effects of mutations or antibiotic treatments to changes in cell shapes supports the idea that the PBP2x and PBP2b class B transpeptidases are associated with septal and p...

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Instability ofackA(Acetate Kinase) Mutations and Their Effects on Acetyl Phosphate and ATP Amounts inStreptococcus pneumoniaeD39

Cited by 49 publications

References 53 publications

Bright Fluorescent Streptococcus pneumoniae for Live-Cell Imaging of Host-Pathogen Interactions

Bright Fluorescent Streptococcus pneumoniae for Live-Cell Imaging of Host-Pathogen Interactions

Acetate Dissimilation and Assimilation in Mycobacterium tuberculosis Depend on Carbon Availability

The Requirement for Pneumococcal MreC and MreD Is Relieved by Inactivation of the Gene Encoding PBP1a

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