Localization of <i>Mycoplasma pneumoniae</i> cytadherence‐associated protein HMW2 by fusion with green fluorescent protein: implications for attachment organelle structure

Balish, Mitchell F.; Santurri, Ryan T.; Ricci, Alessondra M.; Lee, Kyungok K.; Krause, Duncan C.

doi:10.1046/j.1365-2958.2003.03282.x

Cited by 41 publications

(62 citation statements)

References 48 publications

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“…Accordingly, the shorter EDCs observed in mg218 2 mutants with moderate SAD probably reflect the truncated nature of the N-terminal MG218 derivatives. Thereby, our results provide experimental evidence that the length of the EDC corresponds to the length of MG218 modelled as a fully extended a-helix, as has been addressed previously for M. pneumoniae HMW2 (Balish et al, 2003b). Interestingly, we also identified the existence of multiple C-terminal MG218 derivatives in all mg218 2 mutants as well as in the wild-type strain.…”

Section: Discussionsupporting

confidence: 85%

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Role of Mycoplasma genitalium MG218 and MG317 cytoskeletal proteins in terminal organelle organization, gliding motility and cytadherence

et al. 2008

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The terminal organelle is a differentiated structure that plays a key role in mycoplasma cytadherence and locomotion. For this reason, the analysis of Mycoplasma genitalium mutants displaying anomalous terminal organelles could improve our knowledge regarding the structural elements required for proper locomotion. In this study, we isolated several M. genitalium mutants having transposon insertions within the mg218 or mg317 genes, which encode the orthologues of Mycoplasma pneumoniae HMW2 and HMW3 cytoskeletal proteins, respectively. As expected, mg218 " and mg317 " mutants exhibit a reduced gliding motility, although their ability to attach to solid surfaces was not completely abolished. Interestingly, most of the mg218 " mutants expressed N-terminal MG218 derivatives and showed the presence of short terminal organelles retaining many of the functions displayed by this structure in the wild-type strain, suggesting that the N-terminal region of this protein is an essential element in the architecture of the terminal organelle. Separately, the analysis of mg317 " mutants indicates that MG317 protein is involved in the formation of the terminal button and contributes to anchoring the electron-dense core to the cell membrane. The results presented here clearly show that MG218 and MG317 proteins are implicated in the maintenance of gliding motility and cytadherence in M. genitalium.

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Section: Discussionsupporting

confidence: 85%

“…These studies have indicated that the cytoskeletal protein HMW2 is a major structural element of the EDC (Balish et al, 2003b). In addition, it has been suggested that polymers of HMW3 protein surround the core and the terminal button (TB) in a linear pattern, possibly serving to stabilize this structure (Krause, 1996).…”

Section: Introductionmentioning

confidence: 99%

Role of Mycoplasma genitalium MG218 and MG317 cytoskeletal proteins in terminal organelle organization, gliding motility and cytadherence

et al. 2008

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“…HMW2 is a large protein predicted to form dimeric coiled coils, as well as trimeric coiled coils with P28, also a product of the hmw2 gene (5,21). HMW2 is a component of the attachment organelle and appears to be essential for assembly of the electron-dense core (5), which is absent in the hmw2 mutant I-2 (20,31).…”

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confidence: 99%

HMW1 Is Required for Stability and Localization of HMW2 to the Attachment Organelle of Mycoplasma pneumoniae

et al. 2004

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The cytoskeletal proteins HMW1 and HMW2 are components of the terminal organelle of the cell wall-less bacterium Mycoplasma pneumoniae. HMW1 is required for a tapered, filamentous morphology but exhibits accelerated turnover in the absence of HMW2. Here, we report that a reciprocal dependency exists between HMW1 and HMW2, with HMW2 subject to accelerated turnover with the loss of HMW1. Furthermore, the instability of HMW2 correlated with its failure to localize to the attachment organelle. The C-terminal domain of HMW1 is essential for both function and its accelerated turnover in the absence of HMW2. We constructed HMW1 deletion derivatives lacking portions of this domain and examined each for stability and function. The C-terminal 41 residues were particularly important for proper localization and function in cell morphology and P1 localization, but the entire C-terminal domain was required to stabilize HMW2. The significance of these findings in the context of attachment organelle assembly is considered.The cell wall-less prokaryote Mycoplasma pneumoniae causes tracheobronchitis and primary atypical pneumonia in humans. Mycoplasma adherence to host respiratory epithelium (cytadherence) is mediated largely by the attachment organelle, a polar, membrane-bound, differentiated cell extension that is defined by an electron-dense core (7, 15). Extraction of M. pneumoniae cells with the nonionic detergent Triton X-100 (TX) yields a TX-insoluble fraction that includes this core structure and consists largely of cytoskeletal proteins such as HMW1, HMW2, HMW3, and P65 (Table 1) (6,11,13,20,30,36). These proteins contribute to the architecture of the attachment organelle, including the localization of the major adhesin protein P1 to this structure, but little is known regarding their specific roles or the dynamics of their assembly (19).HMW1 is a peripheral membrane protein on the mycoplasma cell surface but lacks typical secretion signal or transmembrane sequences or significant homology to any proteins found beyond the mycoplasmas (2, 3, 9). HMW1 has been localized to the attachment organelle (30, 32) as well as the trailing filament (32), depending upon the experimental approach. Cytadherence mutant M6 (Table 1) lacks HMW1 and produces the truncated cytadherence-associated protein P30 due to an internal, in-frame deletion (22). This mutant has a striking morphology, lacking the tapered filamentous appearance characteristic of wild-type cells, and fails to localize P1 to the attachment organelle (13). Recombinant wild-type HMW1, but not a truncated derivative lacking the C-terminal 112 amino acids, restores a near-normal morphology and P1 clustering to mutant M6 (13), indicating that HMW1 contributes to the architecture of the attachment organelle and that the C-terminal domain of HMW1 is essential for normal function.HMW2 is a large protein predicted to form dimeric coiled coils, as well as trimeric coiled coils with P28, also a product of the hmw2 gene (5, 21). HMW2 is a component of the attachment organelle and app...

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“…M. genitalium is not an ideal model organism; it is a small organism with a long doubling time, has few known genetic tools, and no known defined medium. However, Mycoplasma have been successfully engineered in prior work-for instance, with functioning GFP and TetR proteins 40,41 and on a much larger genome-wide scale in the recent work of Gibson et al 42 This study has focused on one side of the relationship between a synthetic circuit and the host-the effect of the synthetic circuit on the host. We have yet not examined the other side of the relationship-the effect of host processes on the behavior of the synthetic circuit.…”

Section: Future Outlookmentioning

confidence: 99%

Towards a whole-cell modeling approach for synthetic biology

Purcell

Jain

Karr

et al. 2013

Chaos: An Interdisciplinary Journal of Nonlinear Science

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Despite rapid advances over the last decade, synthetic biology lacks the predictive tools needed to enable rational design. Unlike established engineering disciplines, the engineering of synthetic gene circuits still relies heavily on experimental trial-and-error, a time-consuming and inefficient process that slows down the biological design cycle. This reliance on experimental tuning is because current modeling approaches are unable to make reliable predictions about the in vivo behavior of synthetic circuits. A major reason for this lack of predictability is that current models view circuits in isolation, ignoring the vast number of complex cellular processes that impinge on the dynamics of the synthetic circuit and vice versa. To address this problem, we present a modeling approach for the design of synthetic circuits in the context of cellular networks. Using the recently published whole-cell model of Mycoplasma genitalium, we examined the effect of adding genes into the host genome. We also investigated how codon usage correlates with gene expression and find agreement with existing experimental results. Finally, we successfully implemented a synthetic Goodwin oscillator in the whole-cell model. We provide an updated software framework for the whole-cell model that lays the foundation for the integration of wholecell models with synthetic gene circuit models. This software framework is made freely available to the community to enable future extensions. We envision that this approach will be critical to transforming the field of synthetic biology into a rational and predictive engineering discipline. Synthetic biology aims to engineer synthetic genetic circuits to endow cells and organisms with the ability to address new applications, including the production of drugs and industrial products, the design of new therapeutics for human diseases, and the study of basic biological processes. Despite significant advances in the field over the last decade, the synthetic biology design cycle has been hampered by the lack of predictive models. In contrast, more established engineering disciplines, such as civil and electrical engineering can rely on rational design by using models that can capture the behavior of real-world systems with a high degree of accuracy. This is currently not the case for synthetic biology, as models are generally only able to make qualitative predictions about system behavior. As a result, producing a functional engineered biological system usually requires extensive manual tuning by trial-and-error, a timeconsuming process that significantly slows the biological design process. The lack of predictive power in current models is partly because these models account for only the synthetic circuits themselves, but not other ongoing processes in the cells in which they reside. However, the complex dynamics of synthetic circuits may affect the host cell and vice versa, leading to divergence between current models and experimental results. Recently, a whole-cell model of a small and simple bacteri...

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Localization of Mycoplasma pneumoniae cytadherence‐associated protein HMW2 by fusion with green fluorescent protein: implications for attachment organelle structure

Cited by 41 publications

References 48 publications

Role of Mycoplasma genitalium MG218 and MG317 cytoskeletal proteins in terminal organelle organization, gliding motility and cytadherence

Role of Mycoplasma genitalium MG218 and MG317 cytoskeletal proteins in terminal organelle organization, gliding motility and cytadherence

HMW1 Is Required for Stability and Localization of HMW2 to the Attachment Organelle of Mycoplasma pneumoniae

Towards a whole-cell modeling approach for synthetic biology

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