EstG is a novel esterase required for cell envelope integrity

Proper regulation of the bacterial cell envelope is critical for cell survival. Identification and characterization of enzymes that maintain cell envelope homeostasis is crucial, as they can be targets for effective antibiotics. In this study, we have identified a novel enzyme, called EstG, whose activity protects cells from a variety of lethal assaults in the ⍺-proteobacterium Caulobacter crescentus. Despite homology to transpeptidase family cell wall enzymes and an ability to protect against cell wall-targeting antibiotics, EstG does not demonstrate biochemical activity towards cell wall substrates. Instead, EstG is genetically connected to the periplasmic enzymes OpgH and BglX, responsible for synthesis and hydrolysis of osmoregulated periplasmic glucans (OPGs), respectively. The crystal structure of EstG revealed similarities to esterases and transesterases, and we demonstrated esterase activity of EstG in vitro. Using biochemical fractionation, we identified a cyclic hexamer of glucose as a likely substrate of EstG. This molecule is the first OPG described in Caulobacter and establishes a novel class of OPGs, the regulation and modification of which is important for stress survival and adaptation to fluctuating environments. Our data indicate that EstG, BglX, and OpgH comprise a previously unknown OPG pathway in Caulobacter. Ultimately, we propose that EstG is a novel enzyme that, instead of acting on the cell wall, acts on cyclic OPGs to provide resistance to a variety of cellular stresses.


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The bacterial cell envelope is a multi-component structure that protects bacteria from the external 44 environment. The envelope is an essential physical barrier to the surroundings, and the factors 45 responsible for building and maintaining the envelope are therefore ideal targets for antibiotics.

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The Gram-negative cell envelope consists of the inner and outer membranes, with the periplasmic identify genes that were synthetically lethal with ∆CTL expression, using the following strains: wild 124 type (WT), RelA'-producing, and RelA'-producing with ∆CTL. Notably, we identified a gene, 125 CCNA_01638 (hereafter named estG for Esterase for Stress Tolerance acting on Glucans, for 126 reasons described below), that appeared to be essential only in the presence of ∆CTL stress 127 ( Figure 1B). estG acquired abundant transposon insertions in WT and RelA' backgrounds, 128 suggesting that it is non-essential in those strains. However, there were almost no transposon 129 insertions in estG in RelA'-producing cells that also produced ∆CTL, indicating an essential 130 function of EstG in the presence of ∆CTL ( Figure 1B). EstG is an uncharacterized protein that is 131 annotated as a β-lactamase family protein in the transpeptidase superfamily, which primarily 132 consists of PG enzymes. We were therefore interested in studying EstG and its relationship to Since ∆CCNA_01639 had no detectable phenotype or genetic relationship to estG, we focused the remainder of our study on characterizing EstG.

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The classification of EstG as a β-lactamase family protein as well as the hypersensitivity of ∆estG 187 to ∆CTL and PG-targeting antibiotics suggested that EstG might act as a β-lactamase. However, 188 purified EstG displayed negligible activity against nitrocefin, a substrate used to detect β-

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To search for the molecular function of EstG in an unbiased fashion, we isolated and 207 characterized spontaneous suppressors of the ampicillin sensitivity of ∆estG. We sequenced four 208 suppressors total (Supplemental Table 1), but were most intrigued by a suppressing mutation in 209 the essential gene, opgH, a periplasmic glucan glucosyltransferase (OpgHL480P) ( Figure 3A).

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OpgH has been characterized in other organisms as the synthase of osmoregulated periplasmic

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To characterize the suppressing mutation in opgH, we generated the suppressing mutation 218 (opgHL480P) in a clean genetic background, in the presence or absence of estG. In the absence of 219 stress, opgHL480P did not impact growth, but did restore ∆estG cells to a WT colony size ( Figure   220 3A). In the presence of ampicillin, opgHL480P completely restored growth in a ∆estG background 221 ( Figure 3A). We also note that the opgHL480P mutation in a WT background exhibited moderate 222 growth defects in the presence of ampicillin.

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We hypothesized that the opgHL480P mutation might result in a loss of function variant, as the

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Interestingly, in revisiting our original ∆CTL suppressors, we discovered an independent 236 suppressing mutation in opgH that restored growth in the presence of ∆CTL (Supplemental Table   237 1). This mutant, OpgHL434P ( Figure 1A), is also a leucine to proline mutation and is located at the To gain further insight into which pathway(s) EstG may impact, we examined estG on the Fitness 245 Browser database (Wetmore et al., 2015). This database includes sensitivities of a genome-wide 246 library of transposon mutants in Caulobacter to numerous stress and environmental conditions 247 and reports on each gene's mutant fitness profile. This resource reflected ∆estG's sensitivities to 248 cell wall antibiotics and also revealed genes that share a similar sensitivity profile to ∆estG when 249 disrupted (i.e., genes that are "co-fit"). The top hit for co-fitness with estG was an uncharacterized

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Overproduction of BglX in a ∆estG background completely rescued the β-lactam sensitivity of 275 ∆estG ( Figure 4D). Surprisingly, the reverse was not true-overproduction of EstG did not 276 compensate for loss of ∆bglX, which was still sensitive to ampicillin ( Figure 4D). Therefore, though there is a genetic interaction between estG and bglX, these results suggest that EstG and BglX    In some organisms, increased OPG production is thought to compensate for a decrease in 300 environmental osmolarity. In low osmolarity media, OPGs in E. coli comprise up to 5% of the dry 301 weight, while in high osmolarity media, OPGs account for as low as 0.5% of the dry weight 302 (Bontemps-Gallo et al., 2017). With our hypothesis that ∆estG and ∆bglX are defective at some 303 point in the OPG pathway, we altered media osmolarity to assess reliance on OPGs in our 304 mutants. We tested this by adding solutes to the media to increase the osmolarity, which we 305 predicted would alleviate the sensitivities of ∆estG and ∆bglX. When grown in complex media ampicillin is supplemented with 50 mM Tris-HCl to increase the osmolarity ( Figure 4E). The 309 change in osmolarity does not rescue all mutants with ampicillin sensitivity, as we do not see 310 rescue for a strain bearing deletion of the primary b-lactamase, blaA (West et al., 2002). We see  To obtain more insight into a putative substrate for EstG, we determined its structure to 2.1 Å 317 resolution using X-ray crystallography ( Figure Table 2). EstG is annotated as a member of the transpeptidase superfamily, and within this family 320 are the well-studied PG enzymes with an α/β hydrolase fold, such as penicillin binding proteins

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EstG significantly hydrolyzed pNB as compared to the negative control, GST ( Figure 5D). We 364 sought to create a catalytically dead mutant of EstG by mutating the predicted active site serine, vitro, confirming that it is a catalytically dead variant ( Figure 5D). Additionally, when Ser101 is 367 mutated to alanine (S101A) in the chromosomal copy of estG, this mutant phenocopies the β-   Figure 6A). Given that E. coli OPGs are between 1 to 10 kDa, we 378 hypothesized that Caulobacter OPGs might be of similar size. Therefore, we further fractionated to isolate only components within our desired size range. The remaining sample was boiled to 380 remove contaminating proteins, leaving sugars or other heat-resistant metabolites intact. In vitro, 381 we combined this 1-10 kDa periplast isolate with purified EstG or the catalytically dead mutant,

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EstGS101A. We then separated molecules in the treated periplast by high-performance liquid 383 chromatography (HPLC) and selected for peaks that decreased in abundance when mixed with 384 EstG, but not when mixed with EstGS101A. Peaks of interest were then identified by mass 385 spectrometry. Using this approach, we identified a molecule that decreased in abundance ~40% 386 when incubated with EstG ( Figure 6B), indicating that EstG enzymatically modified this substrate 387 in some way. The mass of the parental ion led us to hypothesize that the molecule resembled a-388 cyclodextrin (a-CD), a cyclic, hexameric glucose polymer. Notably, the MS/MS spectra for this 389 molecule in the periplast + EstGS101A (top half of Figure 6C), most closely matches the library 390 spectra for a-CD (bottom half of Figure 6C). Greater than 80% of the fragmentation signal 391 generated from our experiments match the ion profile for a-CD. We next attempted to detect 392 chemical modification of a-CD by EstG using our periplast and mass spectrometry workflow.

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However, due to the complexity of the periplast fraction and the small expected amount of 394 modified a-CD, we were not able to identify a modified a-CD molecule or determine a specific 395 activity of EstG on a-CD. Though this small, cyclic sugar is a novel structure for an OPG, it is

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We next sought to validate a-CD as an EstG substrate in vitro. If a-CD is a substrate for EstG, 401 we reasoned we could add a-CD to the pNB hydrolysis assay and inhibit pNB hydrolysis through

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In this study, we originally set out to identify mechanisms of ∆CTL suppression. We were surprised 433 to primarily recover suppressing mutations in stress response pathways, instead of cell envelope-434 or cell wall-related genes. Activation of stress response pathways typically leads to sweeping 435 changes in cellular physiology, suggesting that the stress imposed by ∆CTL is multifaceted and 436 cannot easily be suppressed by mutation of a single factor. We leveraged (p)ppGpp-mediated 437 suppression of ∆CTL to identify more direct factors involved in surviving ∆CTL-induced stress 438 and, through this approach, found estG. While following up on the role of EstG in (p)ppGpp-439 dependent suppression of ∆CTL, we found that estG is unrelated to (p)ppGpp. Instead, it was the 440 additional antibiotic stress (e.g., introduction of gentamycin marked relA' to produce high 441 (p)ppGpp) in the presence of ∆CTL stress that made estG essential (data not shown). We further 442 confirmed this by deleting estG in a ∆CTL background suppressed by high (p)ppGpp through a 443 hyperactive spoT mutant, which was not lethal (data not shown). In retrospect, this finding is not

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Muramidase reactions were reduced and adjusted to pH 3.5 as explained before. Both soluble 758 and muramidase digested samples were run in the UPLC using the same PG analysis method 759 described above.

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Relative total PG amounts were calculated by comparison of the total intensities of the 762 chromatograms (total area) from three biological replicas normalized to the same OD600 and

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Caulobacter crescentus CdnL is a non-essential RNA polymerase-binding protein whose

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Cell envelope homeostasis during stress is maintained through the actions of EstG and the a variety of environmental changes and antibiotic stresses (represented by yellow lightning bolt).

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Residual plot of the rate data from Figure 6E Table 3.

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Strains and plasmids used in this study.