TatC2 is important for growth of Acinetobacter baylyi under stress conditions Justin M. Langro1 Megan M. Chamberland2 Celena M. Gwin3 Natalia Prakash4 Danielle T. Velez5 Nathan W. Rigel6 1.Department of Biology, Hofstra University, Long Island, New York City, USA 3. Present address: Department of Microbial Pathogenesis, Yale University, New Haven, CT, USA 5. Present Address: Regeneraon Pharmaceuticals, Tarrytown, NY, USA Copyright 2019, Fine Focus all rights reserved Manuscript recieved 8 August 2018; accepted 8 November 2018 TatC in Acinetobacter baylyi• 37 Abstract CORRESPONDING AUTHORS KEYWORDS Department of Biology Hofstra University 318A Gittleson Hall Hempstead, NY 11549 nathan.w.rigel@hofstra.edu Phone: 516-463-6542 Fax: 516-463-5112 • Acinetobacter baylyi • cell envelope • protein export • twin-arginine translocase • tatC2 Protein export pathways are important for bacterial physiology among pathogens and non- pathogens alike. This includes the Twin-Arginine Translocation (Tat) pathway, which transports fully folded proteins across the bacterial cytoplasmic membrane. Some Tat substrates are virulence factors, while others are important for cellular processes like peptidoglycan remodeling. Some bacteria encode more than one copy of each Tat component, including the Gram-negative soil isolate Acinetobacter baylyi. One of these Tat pathways is essential for growth, while the other is not. We constructed a loss-of-function mutation to disrupt the non-essential tatC2 gene and assessed its contribution to cell growth under different environmental conditions. While the tatC2 mutant grew well under standard laboratory conditions, it displayed a growth defect and an aberrant cellular morphology when subjected to high temperature stress including an aberrant cellular morphology. Furthermore, increased sensitivities to detergent suggested a compromised cell envelope. Lastly, using an in vitro co-culture system, we demonstrate that the non-essential Tat pathway provides a growth advantage. The findings of this study establish the importance of the non-essential Tat pathway for optimal growth of A. baylyi in stressful environmental conditions. 38 • Fine Focus, Vol 5 2019 Introduction Transport of proteins between mem- brane-bound compartments is a fundamental bio- logical problem faced by all cellular organisms, in- cluding bacteria. Protein transport is carried out by multi-subunit, membrane-spanning molecular ma- chines (12, 23). In Gram-negative bacteria, most ex- ported proteins cross the inner membrane via the Gen- eral Secretion (Sec) pathway. Proteins that take this path must remain unfolded in order to pass through the SecYEG inner membrane translocase. However, some proteins are exported in a fully folded state by using a separate machine called the Twin-Arginine Translocase (Tat) (15). Proteins destined for export via Sec or Tat contain a recognizable tripartite signal peptide composed of a positively charged region at the N-terminus, followed by a hydrophobic region in the middle and a polar region at the end (30). However, there are several key differences between Sec and Tat signal peptides; among them the charged region of ca- nonical Tat signal peptides contains an arginine dipep- tide within a SRRXFLK sequence motif (6, 8). Signal peptides are recognized by the Sec and Tat machinery during the initial stages of protein translocation, and are cleaved by periplasmic signal peptidases upon ex- port (29). The Tat machinery is composed of TatA, TatB, and TatC proteins; some bacteria lack TatB, but in these cases the role of this protein is performed by TatA (17, 22, 45). Other organisms may also encode TatE, which acts as a functional homolog of TatA (39). To achieve substrate transport, the TatB and TatC pro- teins form a subcomplex in the inner membrane that recognizes the Tat-specific signal peptide (1). Recent evidence suggests that TatA protomers are part of the TatBC receptor complex and that after binding signal peptide, additional TatA subunits are recruited to form the translocase (2, 18). The active translocase requires the proton motive force for translocation of the sub- strate across the inner membrane. Upon export of the substrate, the TatA protomers dissociate from the recognition complex and the cycle can begin again. To date, most studies of this protein trans- porter have been performed using Escherichia coli as the model. Recent analyses of bacterial and archaeal genomes reveals that in some organisms, multiple tat homologs are present (29). In fact, there are reports of Gram-negative bacteria with two distinct Tat trans- locases (24, 33). Similarly, there are two distinct Tat translocases in the Gram-positive bacterium Bacillus subtilis, though in this case only TatA and TatC com- ponents are found. In some cases, these extra Tat components are functionally redundant (4), while in other cases they are used to export specific substrates (13). There are few studies of protein transport in or- ganisms with multiple Tat components, and thus the significance of possessing two distinct Tat pathways is not fully appreciated. In order to better character- ize the importance of dual Tat pathways, we used the Gram-negative soil bacterium Acinetobacter baylyi as a model. Scanning the genome of A. baylyi reveals the presence of two distinct Tat pathways. Remarkably, comprehensive mutagenesis of A. baylyi suggests that the components of one Tat machine are essential for growth, while the compo- nents of the other machine are dispensable (11). For clarity, henceforth we refer to the essential Tat genes as tatA1, tatB1, and tatC1 and the non-essential Tat genes as tatA2, tatB2, and tatC2. In this report, we describe experiments characterizing the non-essen- tial Tat pathway of A. baylyi. Specifically, we sought to understand how Tat-dependent protein export enables the growth of A. baylyi under different envi- ronmental conditions. We uncovered a role for the non-essential Tat pathway in maintaining normal cell envelope integrity and cellular morphology under stress conditions. Using co-culture experiments, we found that the non-essential Tat pathway provides a growth advantage to A. baylyi in competition with Pseudomonads that also possess two Tat machines. Taken together, our findings provide insight into the function of the non-essential Tat pathway of a model soil microbe. TatC in Acinetobacter baylyi • 39 Materials & Methods Strains and growth conditions. Unless otherwise noted, growth of A. baylyi wild-type strain ADP1 (strain 33305 from the American Type Cul- ture Collection; Manassas, VA) was performed at 30 degrees Celsius. For routine culturing, all bacterial strains used in this study were grown in LB Lennox (10 g Bacto-tryptone, 5 g yeast extract, and 5 g NaCl per liter; Fisher Scientific). For growth curve exper- iments, each strain was grown overnight in LB and then diluted 1:100 in fresh media. These cultures were incubated with intermittent shaking at 200 rpm and optical density (OD) measurements (at 600 nm) were taken every 30 minutes for 8-10 hours. All strains were grown in triplicate. Construction of an A. baylyi tatC2::kan insertion-deletion mutant. The A. baylyi tatC2 mutant was constructed using an overlap-extension PCR strategy described previously (3). Briefly, up- stream and downstream sequences flanking tatC2 (ACIAD0521) were amplified by colony PCR from the A. baylyi wild-type strain ADP1 (Fig. 1A). The upstream flanking sequence was amplified using prim- ers A (5’-TGGAGTATATAAAAATGGC-3’) and B (5’-ATTGTTTTAGTACCGAGCTCCTTG- GGCAGGCATGATGTC-3’). The downstream flanking sequence was amplified using primers C (5’-GCCATTTATTATTTCCTTCGATCCTC- GAAAAACGTAG-3’) and D (5’-GATTACCTTTG- GCATCAAC-3’). A 795 bp kanamycin-resis- tance cassette was amplified from plasmid pIM1445 (gift from Ichiro Matsumura) (28) using primers E (5’-GAGCTCGGTACTAAAACAAT-3’) and F (5’-GAAGGAAATAATAAATGGC-3’). These three products were mixed together in equal molar ratios in a new tube and joined together in a final PCR using the outer-most upstream and downstream flanking prim- ers (A and D), resulting in the ::kan insertion-deletion allele. This PCR product was purified and then used to transform naturally competent wild-type A. baylyi (14). Incorporation of the insertion-deletion allele was con- firmed by PCR analysis and DNA sequencing. Construction of pTatC2 expression plasmids. To perform complementation experiments on the tatC2::kan mutant, the wild-type tatC gene was cloned into the BamHI site of pWH1266 by custom gene synthesis (GenScript, Piscataway, NJ) using sequences from the A. baylyi genome (www.biocyc. org). Plasmids were transformed into A. baylyi as follows. From an overnight culture of wild-type strain ADP1, 0.1 ml of cells was gently mixed with 100 ng of plasmid DNA. The mixture was allowed to incubate at room temperature for 1 hour. To allow outgrowth of any transformed cells, 0.9 ml of LB was added to the mixture followed by overnight incubation at 30°C while shaking. The next day, 0.1 ml of the transfor- mation mixture was plated onto LB-ampicillin agar and grown overnight at 30°C to select transformants. Detergent sensitivity assay by Efficiency of Plat- ing method. Cultures of wild-type, tatC2 mutant, and complemented tatC2 mutant bacteria were grown overnight in LB broth, then diluted in series in fresh LB in a 96 well plate. Using a multi-prong metal rep- licator, approximately 2 °l of each dilution was trans- ferred to the surface LB agar or LB agar supplemented with 2% sodium dodecyl sulfate (SDS) and 1 mM ethylenediaminetetraacetic acid (EDTA). Plates were incubated overnight at 30°C and growth on the plate was assessed the next day. Microscopic examination of cellular morpholo- gy. Wild-type and tatC2 mutant starter cultures were grown at 30°C overnight. The next day, each strain was diluted 1:100 in fresh LB broth in duplicate. One set of strains was grown at 30°C while the duplicates were grown at 42°C until late exponential phase. Ten microliters of each culture was spotted onto a glass slide and the smears were allowed to air dry. Follow- ing heat fixation, the smears were stained with crystal violet and visualized using an Olympus BX41 micro- scope under the 100X objective with immersion oil. Images shown are a representative field from at least three independent experiments (Figure 2). Competitive co-culture experiments. Overnight cultures were used to inoculate flasks containing 50 ml of fresh LB broth for competition assays. The optical density (OD600 nm) of each cul- ture was normalized to obtain a final OD of 0.1 in the flask. Flasks were incubated at 200 rpm on a platform shaker at 30°C for 24 hours. One hundred microliter aliquots were taken from the initial inoculum and at 40 • Fine Focus, Vol 5 2019 the 24-hour time point, diluted in 10-fold series to a final dilution of 10-7. To enumerate colony forming units (CFUs), 0.1 ml aliquots were plated from the dilutions to obtain well-isolated colonies. Individual competition assays were performed in triplicate. Input and output CFU counts were used to determine the competitive index (CI) for each A. baylyi strain using the following formula: CI = (Af/Ai)/(Bf/Bi), where A represents the A. baylyi strain and B represents the competitor strain, with f and i denoting the final and initial CFU counts, respectively. CI values were sub- sequently analyzed using one-way ANOVA. Figure 2. The tatC2 mutant displays abnormal cellular morphology under high temperature stress conditions. Overnight cultures of wild-type (left column) and tatC2 mutant (right column) bacte- ria were diluted 1::100 into fresh LB broth and grown to late exponential phase at 30°C (top row) or 42°C (bottom row). Aliquots from each culture were heat- fixed to a glass slide and stained with crystal violet. Images were captured using the 100X objective. Each tick in the scale bar equals 1 um. Figure 1. Strategy to construct a tatC2::kan insertion-deletion mutant. A) Primers used to generate the upstream (A and B) and downstream (C and D) regions flanking tatC2 are indicated by solid arrows. Primers used to for PCR analysis of KanR recombinants are indicated by dashed arrows. B) Col- ony PCR analysis of wild-type and tatC2::kan strains. PCR products were resolved on a 0.7% agarose gel and visualized by staining with ethidium bromide. Molecular weight (MW) size standards are shown (in kb) for comparison. TatC in Acinetobacter baylyi • 41 Figure 3. Colony morphology of wild-type and A. baylyi tatC2 mutant strains. Wild-type (left) and tatC2 mutant (right) A. baylyi strains were grown overnight in LB broth cultures at 30°C. Then serial dilutions of each strain were plated onto LB agar. The plates were incubated overnight at 30°C prior to imaging. Figure 3. Genome architecture of the tat genes of Acinetobacter spp. Homologs of tatA, tatB, and tatC have light shading. Putative alkaline phosphatase phoD has dark shading. Flanking genes of unknown function are labeled with ACIAD genome reference numbers. 42 • Fine Focus, Vol 5 2019 The genome of Acinetobacter baylyi encodes homologs of the Twin-arginine translocation (Tat) pathway. The genus Acinetobacter has recently drawn increased attention due to the rise of drug resistant isolates of Acinetobacter baumannii (31). In addition, Acinetobacter baylyi is of growing interest as a model Gram-negative organism due to its ease of genetic manipulation and diverse metabolic capabilities (14). While exploring the genome of A. baylyi for evidence of known protein export machines, we located homologs of the Tat pathway. Interestingly, when we examined the A. baylyi genome, we noted two homologs each of tatA, tatB, and tatC (Fig. 3). We found that the TatA homologs have the highest sequence identity to each other at 86%, while the sequence identity of the TatB and TatC homologs is lower scoring 62.3% and 70.8%, respectively (Table 1). In addition to the differences in amino acid sequence, there was also a reported difference in function between these two putative Tat pathways. De Berardinis and colleagues reported that the genes encoding one Tat pathway were essential for growth (tatA1B1C1), while genes encoding the other (tatA2B2C2) were dispensable for growth (11). This finding suggests the possibility that each of these Tat pathways has a different function in secreting proteins out of the cytoplasm. In particular, it could be the case that some Tat substrates are specifically routed through one Tat pathway rather than the other. As precedent, the dual Tat pathways of Bacillus subtilis are known to display substrate specificity (32). To clarify the role played by each Tat pathway in A. baylyi physiology, we focused on the non-essential Tat machine. Given the established role of TatC homologs in substrate recognition (19, 20, 35), we started our analysis by creating a loss-of-function tatC2 mutant strain. Construction and phenotypic analysis of a tatC2 mutant. We constructed an insertion-deletion tatC2 mutant using a previously described method (3). The tatC2::kan mutant allele was transformed into the wild-type strain of A. baylyi ADP1, and kanamycin-resistant colonies were selected on LB agar supplemented with kanamycin. Recombination of each mutant allele into the bacterial chromosome was confirmed by PCR and DNA sequence analysis (Fig. 1). We obtained numerous transformants for the tatC2::kan allele, suggesting that this gene is dispensable for growth under standard laboratory conditions, in agreement with previous findings (11). To begin characterizing these mutants, the mutant cultures were assessed for their growth properties. Colonies of the tatC2 mutant are indistinguishable from wild- type A. baylyi when grown on LB plates at 30°C (Fig. 4). When grown in LB broth at 30°C, the tatC2 mutant exhibited similar growth kinetics as wild- type A. baylyi (Fig. 5A). A similar growth trend was observed when we plated for viable counts (data not shown). In some organisms, loss of a functional Tat pathway has been associated with defects in virulence, while in other organisms tat mutants exhibit aberrant morphological and physiological phenotypes under in vitro stress conditions, including when subject to high salt or detergent concentrations (10, 42, 44). We next tested the ability of the tatC2 mutant to grow when challenged with various environmental stresses. The tatC2 mutant is sensitive to detergents and growth at high temperature. Defects in bacterial protein export machines, including the Tat pathway, are often accompanied by increased sensitivity to toxic small molecules (21, 25, 26, 36). In E. coli, the Tat pathway exports proteins that help maintain the structural integrity of the cell envelope. Failure to export these proteins weakens the cell envelope, thereby increasing its permeability. To determine if the non-essential Tat pathway is important for cell envelope integrity in A. baylyi, we tested the ability of the tatC2 mutant to grow in the presence of the detergent SDS. Using an efficiency of plating assay, we found that growth of the tatC2 mutant is impaired by treatment with detergent (Fig. 6). Importantly, we observed that complementation of the detergent sensitivity phenotype of the tatC2 mutant could be achieved by constitutive expression of tatC2 on a plasmid. Given the increased cell envelope permeability associated with the tatC2 mutant, we wondered if the non-essential Tat machine was important to resist temperature stress. Although the tatC2 mutant grew as well as wild-type at 30°C, its growth was impaired at 42°C (Fig. 5B). At later time points, growth of the tatC2 mutant slowed as compared to wild-type cultures grown at 42°C. This finding is consistent with the the function of Tat in other organisms and suggests that the non-essential Tat pathway of A. baylyi plays a role in cell envelope biogenesis (10). Given the established role of Tat in maintaining normal cellular morphology (7), we next examined the cellular morphology of the tatC2 mutant by microscopy (Fig. 2). We found that the tatC2 mutant cells grown at Results TatC in Acinetobacter baylyi • 43 30°C were indistinguishable from the wild-type cells. Both strains appeared as small coccobacilli, which is characteristic of the Acinetobacter genus (41). At 42°C, cells of wild-type A. baylyi were slightly larger than those grown at 30°C. Cells of the tatC2 mutant were longer and thicker when grown at 42°C. Thus TatC2 is important for maintaining normal cellular morphology of A. baylyi under high temperature stress conditions. The non-essential Tat pathway provides a competitive advantage to A. baylyi grown in co- culture. We showed that TatC2 is important for maintaining normal cellular morphology under stressful growth conditions in the laboratory. This led us to wonder how important the non-essential Tat machine is for A. baylyi to grow in the environment. The soil is home to complex microbial communities; these microbes are often in competition with each other for scarce nutrients. In such an environment, some protein secretion systems are important for nutrient acquisition (5, 27, 37), while others are used to directly inhibit growth of microbial competitors (16, 38, 43). With this in mind, we tested whether the non-essential Tat pathway, and TatC2 in particular, was important for the growth of A. baylyi in a competitive co-culture system. Pseudomonas species are closely related to Acinetobacter species, and often share similar environmental niches. Therefore, we selected a subset of Pseudomonas species for the co- culture experiments. As shown by the competitive index (CI) values in Table 2, all three Pseudomonas strains tested grew significantly better than both wild- type and tatC2 mutant strains of A. baylyi. However, when comparing the relative CI values between wild- type and the tatC2 mutant, we noticed that the tatC2 mutant was significantly impaired when grown in competition with P. putida. In competitions with both P. aeruginosa and P. fluorescens, there was no significant difference in CI between wild-type and the tatC2 mutant. The difference in CI does not appear to be due to a secreted toxin as P. putida grown in spent media from either wild-type A. baylyi or the tatC2 mutant showed no difference in growth compared to control (data not shown). 44 • Fine Focus, Vol 5 2019 TatC in Acinetobacter baylyi • 45 Figure 5. Growth of wild-type and A. baylyi tatC2 mutant strains in liquid culture. Wild-type (●) and tatC2 mutant (▲) A. baylyi strains were grown at 30°C (A) or 42°C (B) in LB broth cultures. Overnight cultures of each strain were diluted 1:100 in fresh LB at the start of the experiment. Optical density (OD600 nm) was measured periodically. Each data point represents the mean of triplicate experiments. Error bars indicate standard deviation from the mean. *, P < 0.05 by one-way ANOVA. 46 • Fine Focus, Vol 5 2019 Figure 6. The A. baylyi tatC2 mutant is sensitive to detergents. The indicated strains were tested for detergent sensitivity using an efficiency of plating assay. Overnight cultures of each strain serially diluted and plated onto LB agar or LB agar supplemented with 2% SDS + 1 mM EDTA. Each plate was incubated overnight at 30°C prior to imaging. Shown is a representative experiment from triplicate trials. TatC in Acinetobacter baylyi • 47 Despite the fundamental nature of protein export systems, the important contributions of these pathways to bacterial physiology and growth is often underappreciated. Since its discovery, much has been learned about the structural features and mechanistic details of how the Tat pathway transports proteins. Yet there are still key questions to be addressed. Specifi- cally, how do organisms with two Tat pathways sort their cargo to the correct translocase? What is the benefit to having two Tat pathways? Using A. baylyi as a model system, we have started exploring these ques- tions in detail. By disrupting the non-essential Tat pathway by mutation of tatC2, we uncovered a role for this machine during stress conditions. When exposed to detergents, viability of the tatC2 mutant was decreased as compared to wild-type. This finding supports a role for the non-essential Tat pathway in maintaining integrity of the bacterial cell envelope. We also found that the A. baylyi tatC2 mutant displays aberrant cellu- lar morphology under high temperature stress. Similar phenotypes have been observed in tat mutants of other bacterial species; notably, cells of E. coli tat mutants form long chains because the cell wall amidases im- portant for proper cell division are Tat substrates (7). Our search of the A. baylyi genome did not reveal ho- mologs to these particular amidases. Given that E. coli only possesses a single Tat machine, it is possible that there is a more diverse collection of Tat substrates in organisms with two Tat machines like A. baylyi. We believe that biochemical methods will be needed to identify the full suite of Tat proteins in A. baylyi. While the role of protein export systems in bacterial pathogenesis has been well-studied, the importance of these machines with respect to growth in polymicrobial communities is poorly understood. By using a simple co-culture assay, we uncovered a role for the non-essential Tat pathway when grown in competition with other bacterial species. Interesting- ly, this phenotype was only evident when A. baylyi was grown in competition with P. putida, an organism that also encodes two distinct Tat translocases. To explain this difference in CI among the Pseudomonas strains, we considered the basic physiology of all three organisms. All three grow at a similar rate and under similar con- ditions. P. aeruginosa is a human pathogen but can also be isolated in environmental samples (40). Neither P. fluorescens nor P. putida are human pathogens, however, both are associated with the plant root microbiome (9, 34). Interestingly, when we examined the genomes of each species for tat gene homologs, we found that P. aeruginosa and P. fluorescens possess genes encoding a single Tat pathway, while P. putida encodes genes for two separate and distinct Tat pathways. 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