Shewanella oneidensis MR-1 mutants deficient in EPS production also have affected pellicle morphology

Megan E. Vermilion

Abstract

Shewanella oneidensis MR-1 is known to form metal-reducing pellicle biofilms. To characterize the roles of proteins of Shewanella oneidensis MR-1 in biofilm formation, pellicle phenotypes resulting from mutations at extracellular polymeric substances (EPS) were investigated. Mutational analysis of Shewanella oneidensis MR-1 identified proteins necessary for pellicle formation. The insights of this research asks for attention to anaerobic pellicles morphology and EPS translation regulation. To better understand the biochemical mechanisms of Shewanella oneidensis MR-1 pellicle formation, mutants with altered EPS were evaluated for their ability to produce biofilms.

Background Information The genus Shewanella consists of gram-negative proteobacteria that are rod-shaped, 2-3 μm in length, and 0.4-0.7 μm in diameter. These facultative anaerobes are found in deep sea, soil and sedimentary habitats in a planktonic state as part of a biofilm or swimming with the aid of a single polar flagellum. In 1988, a group of scientists discovered a species of Shewanella that respires by transferring electrons to manganese. It was named Shewanella oneidensis MR-1 (“manganese reducer”) after the lake in which it was discovered, Lake Oneida. This MR-1 species was the first Shewanella genome to be sequenced and has thus become a model system of the study of the genus. Through further research, it became clear Shewanella oneidensis MR-1 had a way to transfer electrons to metal outside of their cells for respiration. Due to Shewanella oneidensis MR-1’s dissimilatory metal reducing activity, it is capable of utilizing multiple terminal electron acceptors during anaerobic respiration including insoluble metal oxides. Scientists have begun to deeply investigate this process. Respiring anaerobically, Shewanella oneidensis MR-1 utilizes substrate-level phosphorylation as a primary energy conservation mechanism to sustain growth. Major organic electron donors for this particular bacterium are formate, lactate and pyruvate19. The impact of Fe(III) reduction in soil and sediment is profound. Ferrous iron is a strong reductant for a

variety of compounds including U(VI). The mineralized product of Fe(III) reduction, magnetite, helps immobilize U(VI) through incorporation into the magnetite20. The process of metal reduction may be dependent on the formation of a biofilm. Multilayer bacterial biofilms may form on the internal or external surfaces of another organism, an abiotic environmental surface, a solid surface substrate such as Fe(III) oxide or at the air-water interface2. In research, biofilms grown as pellicles at the air and liquid medium interface are articulated by three stages: cells touch and attach to each other using flagella and curli fibers; an initial layer of cells produces extracellular polymeric substance (EPS) at the air-liquid interface; and a complex three-dimensional shape matures9,10 The EPS is an important component of biofilms. It is produced by Shewanella oneidensis MR-1 and other biofilm-forming bacteria and is composed of polysaccharides, nucleic acids and various proteins. Cells embedded in a biofilm are unable to chemotaxis for respiration. The synthesis of the EPS essential for the life of immobile cells and is regulated by complex pathways. Comparative gene studies aimed at localizing genes coding for EPS and outer membrane proteins have provided insights to the protein regulation and composition of biofilms5. During the study of bacterial biofilms, a number of genes contributing to EPS and biofilm matrix were studied. Structural units of the biofilm matrix were identified such as curli and flagella. Genes that code for EPS were discovered with the complete genome of Shewanella. Analysis of their function, size and location in the Shewanella oneidensis MR-1 genome was facilitated by a great number of scientists7, 14. Curli fibers, pilus-like structures extending outward from the bacterium, belong to a group of bacterial fibers known as amyloids and are known to play a role in the physiological and pathogenic processes of Escherichia coli and Salmonella25. Curli fibers confer adhesive properties to the bacterium and contribute to biofilm formation by mediating cell-substratum and cell-to-cell contacts11. Expression of two csg operons is required for production of curli polymers in Shewanella oneidensis MR-112. The csgBA operon produces the major and minor structural subunits for curli fibers. Interestingly, during a process called interbacterial complementation, a csgB mutant cell can secrete soluble CsgA that can be assembled on the surface of a cell expressing only csgB. Interbacterial complementation can work when strains are grown within a few millimeters of each other, such as within a biofilm25. Shewanella oneidensis MR-1 have one polar flagella. It is composed of a motor and two homologous flagellin fibers covered in a proteinaceous sheath. The flagella extends away from the bacteria up to ten times the width of its outer membrane. The flagellin subunits are encoded by the fliCDEF operon7, 14. A fully functional flagella will propel the bacteria and make the first contact with substrates and other cells. It is well know that flagella play a vital role in biofilms. They are suspected of signaling EPS production and providing structure to the biofilm matrix16.

Bacteria are able to survive in a remarkably diverse collection of environments. Some of the most overrepresented proteins found in the microbial community of S. oneidensis involve regulatory mechanisms for physiological responses to environmental stimuli2. PepT is a gene from Shewanella with a VanY protein domain and it is vital to bacterial wall synthesis and antibiotic resistance29. The existence of antibiotic resistance in nature suggests that it is not completely a defensive mechanism. Antibiotics may provide a mechanism for selectively responding to environmental stimuli32. One study identifies PepT as a biofilm signal factor using a proteolysis and peptidolysis to regulate the production of EPS26. The methodology for comprehensive profiling of the microbial proteome while confirming the expression of a large number of hypothetical genes is crucial to creating a better understanding of the pathways for metabolism. Metabolism of essential amino acids is necessary to survive. Shewanella oneidensis MR-1’s metabolic pathways are being studied using liquid chromatograpy-mass spectrometry (LC-MS)12. Proteins involved in EPS production require the function of the glutamine-hydrolyzing asparagine synthase enzyme for exopolysaccharide transport, polysaccharide biosynthesis enzymes and bacterial cell wall biosynthesis7. A test for metabolism of S. oneidensis MR-1 with resazurin was developed to quantify bacterial growth rates, and calculate CFU. The resazurin assay proved especially useful for identifying viable cells. The measure of optical density (OD) at 600 nm is indicative of the growth phase. An OD of 0.8 is ideal as it represents the exponential growth phase of the bacteria. Previous studies measure OD of bacteria to measure CFU in response to environmental stimuli or growth conditions.

Materials and Methods Chemicals and media S. oneidensis MR-1 wild-type and the various mutants used in this study were routinely cultured at 26°C in dextrose-free tryptic soy broth (TSB) and Lysogeny Broth (LB). The media for PepT-, Wzz-, Wza-, and FliC- mutants was supplemented with kanamycin at 50 μg/mL1. S. oneidensis MR-1 growth Bacterial strains and media used in this study are summarized in Table 1. CASO cultures (9 mL) were grown aerobically for 16 h at 26°C in a Thermo Incubated Orbital Shaker at 150 rpm. Wild-type and the mutants used were also prepared micro-aerobically at room temperature in non-shaking conditions. The optical density (OD) of S. oneidensis MR-1 cultures was captured at 573 nm by a Beckman DU530 UV/Vis spectrophotometer. Total growth of S. oneidensis MR-1 was analyzed spectrophotometrically within 48 hours. A standard curve of colony forming units (CFU) was created from stepwise dilutions of wild-type S. oneidensis MR-1. CFU measurements of experimental samples were extrapolated from the standard curve. Pellicle formation Strains were grown to OD of 0.8 before 1:500 transfer in LB media to sterile petri dishes. The length of time to reach an OD of 0.8 is shown in Table 2. Plates were incubated at 26°C in non-shaking conditions in LB. Pellicles were observed for a course of five days. Different growth phases observed include: initial surface attachment, monolayer formation, migration to form multilayered microcolonies, production of extracellular matrix, and biofilm maturation with characteristic three-dimensional architecture5. Pictures were taken at various intervals for phenotype observation, see Figure 2 & 3. Metabolism LB cultures (9 mL) were inoculated with S. oneidensis MR-1 wild type and mutants and incubated at 26°C for 1 week in non-shaking lab bench conditions. Cultures were prepared in 12-well cell culture plates. To test for the natural antibiotic resistance of wild-type and the mutants used including WbpH-, cultures were grown with ampicillin at 5% w/v in chemically defined Shewanella growth medium (modified M1) that contained 3 mM PIPES, 7.5 mM NaOH, 28.04 mM NH4Cl, 4.35 mM NaH2PO4, 1.34 mM KCl, 0.05 mM Fe(III)-nitrilotriacetic acid (NTA), 0.68 mM CaCl2. The bacteria were allowed to form biofilms for one week. Cultures were then supplemented with resazurin at 10% v/v. Plates were observed for the resazurin to resorufin oxidation which is visibly confirmed by the blue-to-pink color change in the cultures. After two hours, the optical density of the cultures was measured at 573 nm.

Results The mutant pellicles in this study were compared to wild-type pellicles to observe differences in incubation time and morphology. The thickness and strength of pellicles were affected by only some of the mutations tested in this study. The mutant pellicle phenotypes are listed in Table 2. One of the intriguing results of this study was the finding that cultures grown below freezing temperatures were able to form biofilms although cultures incubated at temperatures over 30°C were unable to form biofilms. The cultures grown in shaking conditions produced the largest pellicles. To increase the dissolved oxygen, we created a microaerobic condition in the Thermo Orbital Shaker for the overnight cultures. Another interesting finding was the natural resistance to ampicillin was decreased in polysaccharide production (WzzB- and Wza-) and cell wall biosynthesis (WbpH-) mutants. This was quantified by the CFU calculated during the resazurin assay in Table 3. The introduction of ampicillin to WbpH- and Wzz- caused a 42% reduction in colony formation. Also, WzzB- mutants were affected by ampicillin, cultures reduced by 14% once introduced to the antibiotic. These results show that wza, wzz and wbp are involved in the mechanism for ampicillin resistance. We focused next on the gene csg to better understand the role of curli fibers in pellicle formation. Our results showed that in addition to loss of curlin, a CsgBA (SO0865-66) deficiency also leads to weak pellicles. The translation of csgBA was shown to be an important component of biofilm morphology. The csgBA operon constitutes the major and minor subunits of curli fibers on the extracellular surface of S. oneidensis MR-1. Since these are the only genes that code for curlin, the pMiniHimar RB1 transposon insertion at SO0865 and SO0866 was expected to result in a complete loss of curli fibers. This was confirmed by the inability of S. oneidensis MR-1 to adhere to substratum and other cells. Unsurprisingly, CsgBA- cells result in flatter colonies and a weaker pellicle. A picture was taken of the biofilm within 24 hours of formation and is displayed in Figure 2. The curli fibers contribute greatly to the biofilm matrix integrity in S. oneidensis MR-1 pellicles. In a similar functional category, we wanted to test the effect on pellicles from the loss of flagella. The gene operon for flagella includes the protein product FliC (SO3237) which is translated into one of the homologous flagellin of which flagella is composed. The FliC- were unsurprisingly unable to form pellicles. Flagella have already been implicated as a vital component for initial cell-substratum contact and cell-to-cell contact. The interesting finding of this study was that FliC- mutants continue to produce EPS despite lacking a biofilm matrix. This shows that the flagella is not solely responsible for activating EPS production or detecting an ideal location for biofilm formation. The loss of D-alanyl-D-alanine carboxypeptidase resulted in an inability to form pellicles. PepT- cells mostly collected below the surface of the media. The pepT gene encodes the enzyme necessary for the synthesis of peptidoglycan and some antibiotic resistances.

It confers VanA and VanB-type glycopeptide resistance by production of cell-wall precursors ending in D-Ala-D-Lac or D-Ala-D-Ser with low binding affinity to vancomycin and N-terminal peptide lysis of high-affinity precursors ending in D-Ala-D-Ala30. The genes in EPS regions were observed by comparing the EPS mutants to Shewanella oneidensis MR-1 wild-type. The genes studied included outer membrane protein W (ompW), aminotransferase (asnB), and polysaccharide biosynthesis proteins (wzz and wza). We observed the mutant pellicles of AsnB- to be thinner and slower to form compared to wild-type using OD measurements recorded in Table 2. The mutants WzzB- and Wza- were unable to form pellicles. The OmpW- mutant formed a thin pellicle with erratic distribution of cells and EPS. The function of these genes was made clearer through conserved domain searches and exploration of gene function in potential orthologs. S. oneidensis MR-1 biofilms were assessed for viability by the irreversible reduction of resazurin to resorufin. The reduction of resazurin is only possible through the oxidation of cytochrome c. The mechanism for metal reduction and cytochrome oxidation is outlined in Figure 1. The change of resazurin to resorufin is observable by the color change of blue to pink3. S. oneidensis MR-1 mutants which failed to reduce resazurin were noted by differences in CFU in Table 3. Triplicate runs were used to calculate CFU estimations. Using Kaleidagraph, the growth curve of S. oneidensis MR-1 was interpolated through the standardized absorbance measurements.

Conclusions A key challenge facing researchers investigating microbial biofilm formation is to elucidate the molecular details of the assembly and formation process. The S. oneidensis MR-1 mutants unable to form pellicles were investigated ab initio. Tools for proteomic analysis included annotated genomic data for S. oneidensis MR-1 on NCBI and published genomic studies5. A comparison of the proposed function of mutated genes and the pellicle phenotype assisted in distinguishing integral components of pellicle formation. S. oneidensis MR-1 metal reduction is dependent on biofilm and EPS components. The matrix of EPS is needed to support embedded cells. EPS mutants resulting in a failure to form a pellicle provided insights to the specific proteins regulating pellicle formation. The rate and magnitude at which bacteria formed pellicles was altered differently with each mutation. Proteins associated with biofilms like curlin subunits have been identified in other biofilm-forming bacteria such as Escherichia coli, Vibrio cholerae, Bifidobacterium animalis and Bacillus subtilis5,6. Adhesion to inert surfaces and development of multilayered cell clusters is a precursor to biofilms. The existence of curli orthologs was first identified in biofilm-forming bacteria Escherichia coli and others were discovered in many other species including Bacillus subtilis and Bifidobacterium animalis. The absence of a curli leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae17. Lack of motility in Escherichia coli led to flatter microcolonies and less biofilm in terms of thickness16. Its importance to biofilm formation is further supported by the results from this study. CsgBA- mutants formed components of the EPS under optimal pellicle growth conditions but the mutants were unable to synthesize curli fibers and made weak pellicles that were easily disrupted, suggesting that curli fimbriae are likely to be a structural component of the pellicles in the biofilm matrix. Questions still remain as to which gene products are utilized for biofilm formation. One such hypothetical putative protein found in Shewanella oneidensis MR-1 is an outer membrane (OM) protein OmpW. Recent research has attempted to implicate OmpW in electron transfer systems for metal reduction but evidence thus far has only showed that mutants deficient in OmpW affect microbial fuel cell production26. Other studies have shown that an OmpW deficiency can lead to altered EPS composition25. In Escherichia coli, the structure of OmpW was observed to be an 8-stranded beta-barrel with a long and narrow hydrophobic channel. Single channel conductance experiments showed that OmpW functions as an ion channel in planar lipid bilayers. Data from crystal structure studies on Escherichia coli and Salmonella typhimurium suggest that members of the OmpW family could be involved in the transport of small hydrophobic molecules across the bacterial outer membrane27, 28. Results from several laboratories indicate that bacterial communication within the biofilm is regulated by a number of OM proteins. The D-alanyl-D-alanine carboxypeptidase coded by pepT is linked with bacterial cell wall production, cell-to-cell communication by interacting with other membrane protein domains and vancomycin resistance. Mutants lacking D,D-carboxypeptidase are unable to form pellicles, see Table 2. Initial production

of EPS components is rumored to regulated by a signaling cascade activated by D,D-carboxypeptidase26. Additionally, the loss of vancomycin resistance in PepT- is caused by the inability to remove high affinity targets (D-ala-D-ala) from the cell wall. Shewanella oneidensis MR-1 has one polar flagella. It is composed of a motor and two homologous flagellin fibers covered in a proteinaceous sheath. The flagella extends away from the bacteria up to ten times the width of its outer membrane. The flagella is able to sense nearby cells and surfaces by changes of resistance when using its motor to rotate16. The flagella is able to send the initial signals that an ideal surface has been found. FliC- mutants unable to synthesize flagellin fibers are also unable to form pellicles. Especially in non-motile conditions, like the pellicles grown for this study, flagella are a strong requirement. Motility influences the biofilm architecture in Escherichia coli as well. Eight Escherichia coli strains were studied in a continuous flow system using confocal microscopy. BW25113 was motility-impaired and formed the worst biofilm. The affected genes included qseB, flhD, fliA, fliC and motA. The gene mutations resulted in flatter biofilms than those formed by DH5alpha16. The flagella mutants from multiple bacterial strains reveals that flagella perform a vital structural function in all biofilms and suggest that the initial signal to produce EPS is controlled by genes for the flagellar motor or some other process. A mutant with a transposon insertion in asnB (SO3175), which is predicted to encode a class II type B asparagine synthase, was recently reported to have pellicles with deficiency in EPS which contained lipids of different composition from wild-type Shewanella oneidensis MR-1. Asparagine synthetase B catalyses the ATP-dependent conversion of aspartate to asparagine. Its protein domains are conserved across many species of bacteria. The aminotransferase domain of AsnB is implicated in exopolysaccharide-associated protein sorting33. The pellicles formed from AsnB- are thin and the EPS is visibly and chemically different from wild-type. Additionally, the growth rate of AsnB- is slower than wild-type. To achieve an OD of 0.8, AsnB- required an additional 2 hours of incubation following the first dilution of overnight cultures. The specific conserved residues in AsnB and the results from the AsnB- mutants confirm the involvement of aminotransferase in biofilm formation. The O antigen is the surface polysaccharide side chain of lipopolysaccharide present in gram-negative bacteria31. The O-antigen genes wzzB and wza work cooperatively to synthesize polysaccharides for the EPS during biofilm production. Recent studies of WzzB- biofilms showed little difference in chemical composition from Shewanella oneidensis MR-1 wild-type biofilms. In contrast, the EPS from Wza- biofilms contained increased amounts of macromolecules, specifically phospholipids, proteins, and nucleic acids25. In the biofilm set-up from this study, no pellicles formed from either WzzB- or Wza-. This may be due to a loss of antibiotic resistance conferred by the polysaccharide chain length determinant protein WzzB. The biofilms were prepared in a Thermo Orbital Shaker using 150 rpm and temperature controls to test the effect that shaking cultures and temperature could have on the ability of wild-type Shewanella oneidensis MR-1 to form pellicles below freezing temperatures.

The introduction of oxygen to the cultures was to assist in metabolism. We expected to see an increase in growth rates of Shewanella oneidensis MR-1 in response to higher DO because oxygen is the optimal terminal electron acceptor. The effect of temperature

on biofilm formation was explored from 0 to 34℃ using temperature controls on the Orbital Shaker. It was found that pellicles grew readily from cultures grown from temperature below 30℃. Shewanella oneidensis MR-1 can survive temperatures above

30℃, but pellicles do not form from these cultures. Antibiotics can kill bacteria (bactericidal) or sometimes just nullify growth (bacteriostatic). To understand how antibiotics work, and further, why they stop being effective requires an examination of the targets for the main classes of these antibacterial drugs. There are three targets for the main antibacterial drugs: (1) bacterial cell-wall biosynthesis; (2) bacterial protein synthesis; and (3) bacterial DNA replication and repair. Shewanella oneidensis MR-1 is resistant to a host of antibiotics including vancomycin and ampicillin32. To test for the genes responsible for antibiotic resistances in wild-type Shewanella oneidensis MR-1, the effect of mutations at putative antibiotic drug targets were quantified by CFU. The gene for biosynthesis cell-wall protein WbpH (SO3176) could be connected to antibiotic resistance. The molecules that bind to WbpH on the cellular surface should be investigated to determine the molecular mechanism. Additionally, the mutants in polysaccharide production (Wza- and WzzB-), and cell wall synthesis (WbpH-) were found to be sensitive to Ampicillin, implicating their connection to antibiotic resistance. The purpose of this study was to identify how mutations in putative EPS biosynthesis could affect pellicles. Using information it could be possible to modify the genes necessary for heavy metal reduction to augment biofilms, and thereby enhance a bacterium’s ability to form pellicle. We showed that cell wall biosynthesis proteins, outer membrane proteins, flagella, curli fibers and proteins involved in polysaccharide production had significant roles in pellicle formation.

Acknowledgments This work was supported by Whitman College Biochemistry, Biophysics, and Molecular Biology Department under the direction of Dr. Sara Mae Belchik. We thank Pacific Northwest National Laboratories for Shewanella oneidensis MR-1 wild-type and mutant cultures.

Figures Table 1. Bacterial strains used for this study.

Strains Description and Predicted Protein Function Reference

S. oneidensis

MR-1 wt

Manganese-reducing strain (Lake Oneida, NY). (14)

CsgBA- pMiniHimar RB1 transposon insertion in SO0865-6. Curlin major and minor subunits

This study

OmpW- SO1673 (ompW) deletion derivative of MR-1. Outer membrane protein of unknown function.

(25)

PepT- pMiniHimar RB1 transposon insertion in SO2472. Requires Kma selection. D-alanyl-D-alanine carboxypeptidase

This study

AsnB- pMiniHimar RB1 transposon insertion in SO3175 (asnB). Requires Kma selection. Glutamine hydrolyzing asparagine synthase

(25)

WbpH- pMiniHimar RB1 transposon insertion in SO3176 (wbpH). Requires Kma selection. O-antigen biosynthesis glycosyl transferase family 4.

This study

WzzB- pMiniHimar RB1 transposon insertion in SO3191 (wzzB). Requires Kma selection. O-antigen chain length determinant protein.

(25)

Wza- pMiniHimar RB1 transposon insertion in SO3193 (wza). Requires Kma selection. Polysaccharide biosynthesis protein

(25)

FliC- pMiniHimar RB1 transposon insertion in SO3237 (fliC). Flagella subunit

This study

aKm, kanamycin.

Table 2. Shewanella oneidensis MR-1 wild-type and mutant pellicles.

S. oneidensis Pellicle Thickness Incubation Timea

MR-1 wt Normal 4

CsgBA- Thin Pellicle 4

OmpW- Thin Pellicle 4

PepT- No Pellicle 4

AsnB- Thin Pellicle 6.5

WzzB- No Pellicle 4

Wza- No Pellicle 4

FliC- No Pellicle 4.5

aIndicates average time in hours to achieve an OD of 0.6 following a 1:100 dilution from overnight cultures (CASO)

Table 3. S. oneidensis MR-1 Colony Forming Units.

S. oneidensis CFUa CFUb Reduction of CFU by Ampicillin

MR-1 wt 2.45 x 109 1.87 x 109 2%

CsgA-, B- 2.47 x 109 2.44 x 109 1%

OmpW- 2.47 x 109 2.44 x 109 1%

AsnB- 2.47 x 109 2.44 x 109 1%

WbpH- 2.48 x 109 1.44 x 109 42%

WzzB- 2.48 x 109 1.44 x 109 42%

Wza- 1.75 x 109 1.50 x 109 14%

a Calculated CFU in LB liquid medium 12 well plates, normal growth conditions b Amp is added to fully-formed biofilms at 5% w/v to LB

Figure 1. Proposed Mtr extracellular electron transfer pathway for Fe(III) oxide reduction of S. oneidensis MR-1.

The protein components identified to date for the Mtr pathway include CymA, MtrA, MtrB, MtrC, and OmcA. Together the MtrAB facilitate the electron transfer across the OM to the MtrC and OmcA on the bacterial surface. MtrC and OmcA are the terminal reductases which bind the surface of Fe(III) oxides and transfer electrons directly to the oxides via heme proteins. Flavins are used in the Mtr pathway to increase reaction rates. The flavins are secreted by S. oneidensis MR-1 as diffusible shuttles for Fe(III) oxide reductions. The sizes of the components depicted are not drawn to scale.

Figure 2. A. Pellicle of S. oneidensis MR-1 wild-type

B. Pellicle of CsgBA- mutant with deletion of major and minor curlin subunits

Images were taken of the pellicle after 48 hours of growth in LB. The abundance of colonies and thickness of S. oneidensis MR-1 wild-type pellicles (A) were compared to CsgBA- (B), OmpW-, PepT-, AsnB-, WzzB-, and Wza-. Biofilms were prepared as described in Materials and Methods.

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