Suppressor Analysis Reveals a Role for SecY in the SecA2-Dependent Protein Export Pathway of Mycobacteria

Ligon, Lauren S.; Rigel, Nathan W.; Romanchuk, Artur; Jones, Corbin D.; Braunstein, Miriam

doi:10.1128/jb.00630-13

Cited by 19 publications

(28 citation statements)

References 41 publications

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“…This system has been extensively studied in streptococci and is highly specialized to export glycosylated proteins that are incompatible with the canonical Sec system (13,(17)(18)(19)(20). Other bacteria contain a SecA2-only system, which is thought to interact with the canonical SecYEG channel (16,21,22). The repertoire of proteins that require the accessory SecA2 for secretion is relatively small and varies between organisms (13, 17).…”

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A prl Mutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

2015

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The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogen Listeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence in L. monocytogenes; however, the mechanism of export via this pathway is poorly understood. L. monocytogenes secA2 mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility to L. monocytogenes secA2 mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by the lmo2769 operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation in secY was identified that conferred smooth colony morphology to secA2 mutants, restored wild-type plaque formation, and increased virulence in mice. This secY mutation resembled a prl suppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ⌬secA2prlA1 mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although the prl mutation largely suppressed almost all of the measurable SecA2-dependent traits, the ⌬secA2prlA1 mutant was still less virulent in vivo than the wild-type strain, suggesting that SecA2 function was still required for pathogenesis.T he essential general secretory (Sec) pathway is responsible for exporting the majority of secreted proteins across the bacterial cell membrane (1-3). Much of what we know about this pathway was discovered in Escherichia coli by using genetic screens to identify loss-of-function mutations in components of the Sec system. This led to the identification of components of the SecYEG complex that forms the translocation channel and the SecA ATPase that binds to signal sequences to drive the export of precursor proteins across the channel (3-9). In contrast to loss-of-function sec mutations, prl alleles are gain-of-function mutations, which expand the repertoire of substrates being exported across the SecYEG channel (6-8, 10, 11). The most dominant prl variants are in secY, known as prlA mutations, which allow for the secretion of proteins with altered signal sequence (7, 9, 11) without significantly altering the secretion of other proteins (6,12).In addition to the canonical SecA, an accessory cytosolic ATPase, SecA2, has been identified in Mycobacterium and a subset of other Gram-positive bacteria. Unlike SecA, SecA2 is not essential for cell viability but is required for virulence in some pathogenic bacteria (13-15). SecA1 and SecA2 are homologous but are not interchangeable (16)...

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A prl Mutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

2015

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“…Such phenotypes often result from a dominant-negative protein being locked in a nonproductive complex with its normal binding partners. Furthermore, we showed extragenic suppressors that overexpress SecY suppress the secA2 K129R dominant-negative phenotype, which argues for an interaction between SecA2 K129R and SecY (30). Here, we identified intragenic suppressors of secA2 K129R , and all of them mapped to the surface of the SecA2 structure.…”

Section: Seca2mentioning

confidence: 88%

“…An M. smegmatis strain expressing the dominant-negative MsSecA2 K129R , which has an amino acid substitution in the Walker box (equivalent to K115 in MtbSecA2) was used. All suppressors identified reversed the severe dominant-negative phenotypes caused by SecA2 K129R , as assessed by azide sensitivity assays (5) and colony size on rich agar (30) (Fig. 6 and data not shown), but they still exhibited a phenotype similar to that of a ⌬secA2 null mutant.…”

Section: Seca Is Another Critical Residue In the 2hf (36) Although Imentioning

confidence: 99%

“…Spontaneous suppressors of the secA2 K129R strain were isolated by plating onto Mueller-Hinton agar at 37°C, as described previously (30). The secA2 K129R strain has the chromosomal secA2 gene deleted and carries a copy of the secA2 gene encoding SecA2 K129R integrated at the chromosomal L5 att site.…”

Section: Methodsmentioning

confidence: 99%

“…Furthermore, SecA2 variants lacking ATPase activity due to an amino acid substitution in the Walker box are dominant negative, and a secA2 dominant-negative mutant exhibits secA2 mutant phenotypes (a growth defect on rich agar and azide sensitivity) that are more severe than those exhibited by a ⌬secA2 null mutant (29). Extragenic suppressors of this dominant-negative secA2 allele map to the secY promoter, and increased SecY levels suppress the secA2 dominant-negative phenotype (30). These findings suggest that the SecA2 dominantnegative protein is locked in a nonproductive interaction with the essential SecYEG channel, which inhibits SecYEG function but can be overcome by increased SecY production.…”

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Structural Similarities and Differences between Two Functionally Distinct SecA Proteins, Mycobacterium tuberculosis SecA1 and SecA2

et al. 2016

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While SecA is the ATPase component of the major bacterial secretory (Sec) system, mycobacteria and some Gram-positive pathogens have a second paralog, SecA2. In bacteria with two SecA paralogs, each SecA is functionally distinct, and they cannot compensate for one another. Compared to SecA1, SecA2 exports a distinct and smaller set of substrates, some of which have roles in virulence. In the mycobacterial system, some SecA2-dependent substrates lack a signal peptide, while others contain a signal peptide but possess features in the mature protein that necessitate a role for SecA2 in their export. It is unclear how SecA2 functions in protein export, and one open question is whether SecA2 works with the canonical SecYEG channel to export proteins. In this study, we report the structure of Mycobacterium tuberculosis SecA2 (MtbSecA2), which is the first structure of any SecA2 protein. A high level of structural similarity is observed between SecA2 and SecA1. The major structural difference is the absence of the helical wing domain, which is likely to play a role in how MtbSecA2 recognizes its unique substrates. Importantly, structural features critical to the interaction between SecA1 and SecYEG are preserved in SecA2. Furthermore, suppressor mutations of a dominant-negative secA2 mutant map to the surface of SecA2 and help identify functional regions of SecA2 that may promote interactions with SecYEG or the translocating polypeptide substrate. These results support a model in which the mycobacterial SecA2 works with SecYEG. IMPORTANCESecA2 is a paralog of SecA1, which is the ATPase of the canonical bacterial Sec secretion system. SecA2 has a nonredundant function with SecA1, and SecA2 exports a distinct and smaller set of substrates than SecA1. This work reports the crystal structure of SecA2 of Mycobacterium tuberculosis (the first SecA2 structure reported for any organism). Many of the structural features of SecA1 are conserved in the SecA2 structure, including putative contacts with the SecYEG channel. Several structural differences are also identified that could relate to the unique function and selectivity of SecA2. Suppressor mutations of a secA2 mutant map to the surface of SecA2 and help identify functional regions of SecA2 that may promote interactions with SecYEG.

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The Canonical and Accessory Sec System of Gram-positive Bacteria

Prabudiansyah

Driessen

2016

Current Topics in Microbiology and Immunology

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The Sec system is present in all bacteria and responsible for the translocation of the majority of proteins across the cytoplasmic membrane. The system consists of two principal components: the ATPase motor protein, SecA, and the protein-conducting channel, SecYEG. In addition to this canonical Sec system, several Gram-positive bacteria also possess a so-called accessory Sec system. This is a specialized translocation system that is responsible for the export of a subset of secretory proteins, including virulence factors. The accessory Sec system consists of a second SecA paralog, termed SecA2, with or without a second SecY paralog, termed SecY2. In some bacteria, the accessory Sec system is dependent on the canonical Sec system for functionality, while in other bacteria, they can function independently. In this review, we provide an overview of the current knowledge of the canonical and accessory Sec system of Gram-positive bacteria with a focus on the primary component of the Sec translocase, SecA and SecYEG.

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Suppressor Analysis Reveals a Role for SecY in the SecA2-Dependent Protein Export Pathway of Mycobacteria

Cited by 19 publications

References 41 publications

A prl Mutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

A prl Mutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

Structural Similarities and Differences between Two Functionally Distinct SecA Proteins, Mycobacterium tuberculosis SecA1 and SecA2

The Canonical and Accessory Sec System of Gram-positive Bacteria

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