Cloning and expression in Escherichia coli of the structural gene coding for the monomeric protein of the S layer of Thermus thermophilus HB8

Faraldo, Marisa M.; Pedro, Miguel A. de; Berenguer, José

doi:10.1128/jb.173.17.5346-5351.1991

Cited by 26 publications

(22 citation statements)

References 32 publications

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“…8B, a band corresponding to the wild-type SlpA protein was strongly labeled, while the SlpA.X protein remained undetected. In addition, we also could detect other labeled bands in this strain which most probably corresponded to degradation products and oligomers of SlpA, as has been previously shown (7,9,10). As a negative control of the antiserum specificity, a parallel Western blot was carried out in which the peptidoglycan fraction was not added, resulting in the complete absence of labeling after incubation with the antiserum (data not shown).…”

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A conserved motif in S-layer proteins is involved in peptidoglycan binding in Thermus thermophilus

et al. 1996

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There is experimental evidence to suggest that the 100-kDa S-layer protein from Thermus thermophilus HB8 binds to the peptidoglycan cell wall. This property could be related to the presence of a region ( Paracrystalline surface layers (S-layers) are commonly found within prokaryotes belonging to different phylogenetic groups (22). As the outermost envelope, the functions of S-layers are related to their interaction with the specific environment in which these organisms grow. However, their constant presence in bacteria belonging to the oldest phylogenetic groups and the severe defects shown by S-layer-defective mutants in these groups (13, 17) suggest that primitive S-layers could have played a role essentially related to the maintenance of the cell morphology and envelope integrity (2, 22).Three-dimensional reconstruction of S-layers has revealed features common among them (22), independently of differences in their specific functions. Essentially, S-layers present a smooth surface which faces outside and a more corrugated one that binds them to the underlying envelope, whatever its nature (peptidoglycan, outer membrane, or lipopolysaccharide). At higher resolution, this corrugated side shows up as wide columns, located at the main symmetry axis, that are interconnected through a network of thin contacts at the surface. This network of contacts generates the smooth face of the S-layer.This common morphology of the S-layer is built up by a single protein component that is folded in at least two clear domains: a heavy domain, which interacts with other equivalent domains to form the wide columns that bind the S-layer to the cell, and one or more light domains to connect them at the surface (28). However, the resolution of the three-dimensional models available at present does not allow a correlation between structural topology and protein sequence to be made. In addition, the scarcity of both S-layer models and protein sequences and the high divergence found within the latter do not in general allow association of specific sequence motifs or patterns with specific structural features. However, in a recent article, Lupas et al. (20) have described the existence of one or more copies of a protein domain known as the S-layer homology (SLH) region in a number of S-layers and regular membrane proteins from unrelated bacteria. On the basis of the homology, also found with extracellular enzymes from grampositive bacteria (20), a peptidoglycan-binding function was tentatively assigned to this domain.Despite belonging to a phylogenetic group different from that of gram positive bacteria, the 100-kDa S-layer protein from Thermus thermophilus HB8 was included among those that contain an SLH domain (11,20). In this 917-amino-acidlong protein (SlpA), the SLH domain was found from amino acid positions 28 to 87, thus suggesting the possibility of an interaction in vivo between the S-layer and the peptidoglycan. Two biochemical data can be argued to support the existence of such interactions. First, the solubilization of SlpA w...

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A conserved motif in S-layer proteins is involved in peptidoglycan binding in Thermus thermophilus

et al. 1996

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“…Its coding gene, slpA (for S-layer protein) has been cloned and sequenced (7,8), and its promoter has been found to function in both Thermus spp. and Escherichia coli (8).…”

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Horizontal transference of S-layer genes within Thermus thermophilus

et al. 1995

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The S-layers of Thermus thermophilus HB27 and T. thermophilus HB8 are composed of protein units of 95 kDa (P95) and 100 kDa (P100), respectively. We have selected S-layer deletion mutants from both strains by complete replacement of the slpA gene. Mutants of the two strains showed similar defects in growth and morphology and overproduced an external cell envelope inside of which cells remained after division. However, the nature of this external layer is strain specific, being easily stained and regular in the HB8⌬slpA derivative and amorphous and poorly stained in the HB27⌬slpA strain. The addition of chromosomic DNA from T. thermophilus HB8 to growing cultures of T. thermophilus HB27⌬slpA led to the selection of a new strain, HB27C8, which expressed a functional S-layer composed of the P100 protein. Conversely, the addition of chromosomic DNA from T. thermophilus HB27 to growing cultures of T. thermophilus HB8⌬slpA allowed the isolation of strain HB8C27, which expressed a functional S-layer composed of the P95 protein. The driving force which selected the transference of the S-layer genes in these experiments was the difference in growth rates, one of the main factors leading to selection in natural environments.In many mesophilic bacteria, the presence of crystalline surface layers (S-layers) seems to be a strain-specific character, whose loss upon optimal growth conditions results in S-layer mutants that do not present any apparent phenotypic defect (12, 21). By contrast, most thermophiles, especially those belonging to the oldest phylogenetic branches (31), contain Slayer as an almost universal character, thus suggesting for these structures important roles in cell viability or membrane integrity at high temperatures. In fact, the presence of S-layer in evolutionarily old thermophilic bacteria and their structural simplicity led to the suggestion of an ancient evolutionary origin for such structures (26).As S-layers completely surround the cells, their building units constitute one of the major membrane proteins. Accordingly, synthesis of the S-layer is a metabolically expensive process which could be selected during evolution only as a result of the existence of strong selective pressures. However, little is known about such selective pressures, and even less is known about the role(s) that S-layers could play in mesophilic or thermophilic natural environments.Like any cell surface components, S-layers should be subjected to strong selective pressures, the most important of which could be the presence of hydrolytic enzymes (essentially proteases) and bacteriophages (as binding sites). Such selective pressures could be the factors responsible for the sequence divergence of the S-layer genes (21). In fact, it has been not possible to obtain clear phylogenetic relationships between S-layer genes, even from related organisms. In addition, the possibility exists that horizontal transference of S-layer genes within genetically related strains contributed to the present sequence divergence, in a way similar to th...

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“…Polyclonal rabbit antiserum against the S-layer protein from T. thermophilus HB8 (Faraldo et al, 1991) was used to reveal slpA products. Antisera against Rep6 (this work) and SlpM (Olabarría et al, 1996b) proteins were obtained from New Zealand rabbits using PAGE-purified proteins as antigens, as described (Boulard and Lecroisey, 1982).…”

Section: Western Blottingmentioning

confidence: 99%

“…Thermus thermophilus HB8 is an extreme thermophilic bacterium (Oshima and Imahori, 1974) that has a hexagonal S-layer (Castó n et al, 1988), whose structural unit is encoded by the slpA gene (Faraldo et al, 1991;Faraldo et al, 1992). The deletion of slpA gives rise to severe phenotypic alterations (Lasa et al, 1992;Ferná ndezHerrero et al, 1995b).…”

Section: Introductionmentioning

confidence: 99%

Surface proteins and a novel transcription factor regulate the expression of the S‐layer gene in Thermus thermophilus HB8

Fernández-Herrero

Olabarría

Berenguer

1997

Molecular Microbiology

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SummaryWe have identified proteins that control the expression of slpA, the gene encoding the crystalline surface layer of Thermus thermophilus HB8. We cloned three genes from T. thermophilus that specifically repressed the expression of the slpA promoter in Escherichia coli. The proteins encoded by two of them (Rep6 and Rep29) bound in vitro to the slpA promoter, while that from the third (Rep54) bound specifically to the 5Ј-untranslated region (5ЈUTR) of the slpA mRNA. Rep6 protein was identified as a C-fragment from a Thermus cytoplasmic basic protein of 28 kDa, whose coding gene, slrA (for S-layer regulator), was characterized. Surprisingly, Rep29 was identified as a C-fragment of SlpM, an S-layerlike protein that is overexpressed in slpA mutants. Insertional inactivation of slrA and slpM demonstrated their in vivo function in the control of slpA transcription: SlrA acts as a repressor, and SlpM as an activator. Even more surprising was the identification of Rep54, the 5ЈUTR mRNA-binding protein, as a C-terminal fragment of the SlpA protein. This result, in addition to further in vivo evidence presented here, supports the existence of a translational autoregulation in slpA expression. The physiological meaning of overlapping transcriptional and translational controls of S-layer expression, and its relationships with other systems, are discussed.

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Cloning and expression in Escherichia coli of the structural gene coding for the monomeric protein of the S layer of Thermus thermophilus HB8

Cited by 26 publications

References 32 publications

A conserved motif in S-layer proteins is involved in peptidoglycan binding in Thermus thermophilus

A conserved motif in S-layer proteins is involved in peptidoglycan binding in Thermus thermophilus

Horizontal transference of S-layer genes within Thermus thermophilus

Surface proteins and a novel transcription factor regulate the expression of the S‐layer gene in Thermus thermophilus HB8

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