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Photobacterium profundum SS9 is a γ-proteobacterium which grows optimally at 15°C and 28 MPa (a psychrophilic piezophile) and can grow over a range of temperatures (2-20oC) and pressures (0.1-90 MPa). Previous research had demonstrated that P. profundum SS9 adapts its membrane proteins and phospholipids in response to growth conditions. In this study, methodology was developed for growing P. profundum SS9 under cold temperatures and high pressures in both liquid and solid cultures. The effect of changing growth conditions on cell envelope polysaccharides was then investigated. The lipopolysaccharide (LPS) profile of a rifampicin resistant P. profundum SS9 derivative, SS9R, was shown to change at 0.1 MPa with respect to temperature and at 15°C with respect to pressure. Compositional analysis showed that the LPS was almost entirely composed of glucose. This provides evidence that, under these conditions, the major polysaccharide produced by P. profundum SS9 is a glucan. Two putative polysaccharide mutants, FL26 & FL9, were previously isolated from a screen for cold-sensitive mutants of P. profundum SS9R. Both mutants displayed an increased sensitivity to cold temperatures on solid medium and were unaffected in their growth at high pressure. FL26 was found to exhibit an LPS alteration similar to previously published O-antigen ligase mutants, providing evidence that this mutant is likely to lack O-antigen ligase. Interestingly, FL26 was also shown to have a reduced ability to form biofilms and had increased swimming motility. This suggests that there are a number of changes which occur in FL26 in the absence of O-antigen. FL9 was found to have an altered LPS and capsular polysaccharide (CPS), similar to an E. coli wzc mutant. In E. coli, Wzc is involved in the polymerisation and transport of CPS, disruption of which can also lead to LPS alterations. The LPS and CPS alterations may lead to the cold-sensitivity phenotype, either individually or in combination. In conclusion, alterations in the cell envelope polysaccharides were shown to affect cold temperature sensitivity on solid agar. Cold-sensitivity is most likely directly related to the LPS alterations and stability of the membrane under cold temperatures. Exopolysaccharides (EPS) have previously been shown to affect desiccation and freezethaw resistance, making it is possible that the CPS plays a similar role in this case.

The cell envelope of mycobacteria is critical for the survival and virulence of pathogenic species during infection, and its biosynthesis has been a proven drug target. Therefore, finding new targets in the biosynthetic pathway of cell envelope components is of great interest. Mycobacterium smegmatis is a model organism for the study of the devastating pathogen Mycobacterium tuberculosis. Previously, lipomannan elongation factor A (LmeA) has been identified as a cell envelope protein that is critical for the control of mannan chain length of lipomannan (LM) and lipoarabinomannan (LAM), lipoglycan components of the cell envelope. The deletion mutant, ∆lmeA, accumulates abnormal LM/LAM with fewer mannan residues. To understand the importance of this protein, the antibiotic sensitivity of ∆lmeA was tested using a resazurin-based viability assay. We found that the lmeA deletion leads to increased sensitivities to antibiotics such as vancomycin and erythromycin, and lmeA overexpression leads to increased antibiotic resistance. To directly test if the increased antibiotic sensitivity is due to the defective permeability barrier, we used an ethidium bromide uptake assay and found that ∆lmeA is more efficient in taking up ethidium bromide in the cell. We have also found that LmeA is important for protein stabilization under stress conditions. MptA is an α1,6-mannosyltransferase involved in elongation of LM and LAM mannan chain. During stress conditions in the ΔlmeA mutant, levels of MptA decrease significantly relative to wild-type. This also results in delayed doubling time after stress, a phenotype not seen in this mutant under normal growth conditions. In addition, the ΔlmeA mutant has differential protein expression during stress conditions relative to ΔlmeA in log phase, or to wild-type in either condition. To help elucidate the role of LmeA at the molecular level, binding behavior of this protein to membrane fractions was determined. In a subcellular fractionation analysis, LmeA localizes to fractions containing plasma membrane, which is tightly bound to cell wall layers. To test the binding of LmeA to membrane further, LmeA was heterologously expressed in Escherichia coli, purified, and mixed M. smegmatis cell lysate. LmeA localized to intracellular domain fractions (IMD), indicating that LmeA is capable of localizing to fractions containing only plasma membrane. Consistent with this finding, LmeA is capable of binding to spheroplasts in both an ELISA setting as well as in a sucrose gradient fractionation setting. It has also been determined that ΔlmeA has a defective capsular layer with a unique phenotype relative to other strains. We have concluded that LmeA is important for antibiotic resistance, cell envelope permeability, capsule formation, stress response, and have also determined its binding properties.

Type IV pili (T4P) are protein filaments found on the surface of a variety of bacterial species and mediate biofilm formation, adhesion, and flagellum-independent twitching motility. The biogenesis of T4P is dependent on a cell envelope-spanning, multiprotein complex that localizes to the poles in rod-shaped cells. How these proteins localize and cross the peptidoglycan (PG) layer in the absence of dedicated PG-hydrolyzing enzymes is unknown. In P. aeruginosa, PilMNOP interact to form the alignment subcomplex, connected via PilP to PilQ, which forms the outer membrane secretin. We hypothesized that polar localization and integration of the T4P machinery was driven by ordered recruitment to future sites of cell division, placing assembly system components at division septa in the correct position before daughter-cell separation. To determine which T4P components are essential for localization of the complex, we fused the T4P inner membrane assembly protein PilO to the fluorescent protein mCherry to monitor its localization. mCherry-PilO localized to the cell poles and midcell in wild type bacteria. However, it was delocalized in a strain lacking PilQ. A PilQ-mCherry fusion localized to the cell poles, likely through its putative septal PG binding AmiN domains, suggesting that PilQ binds PG and thus localizes its partners to future sites of cell division. In the absence of the associated pilotin protein (PilF), which is required for PilQ multimerization in the OM, T4P components were polarly localized, implying that localization is not dependent on secretin formation. The results of this research support a pre-installation mechanism for integration of protein complexes in the gram negative cell envelope without PG hydrolysis, which may be applicable to other systems. / Thesis / Master of Science (MSc)

Rhodococcus is an important industrial organism that possesses diverse metabolic capabilities, it also has a unique cell envelope, composed of an outer layer of mycolic acids and glycolipids (free or bound lipids generally linked to the sugar trehalose). Rhodococcus is able to transform nitriles to the corresponding amide by the enzyme Nitrile Hydratase (NHase), therefore rhodococcal cells can be utilized as biocatalysts in the detoxification of nitrile waste water or in the production of industrially important amides such as acrylamide. However, the NHase within the native cells must be stable with high activity. This research examined how NHase activity and stability can be increased in native cells by changing growth media composition, the impact on the rhodococcal cell envelope was also studied. Growth media composition was altered by supplementing different sugars such as fructose, maltose or maltodextrin to replace glucose in rich solid media containing cobalt and urea for induction of NHase. The supplementation of maltose or maltodextrin resulted in significantly higher NHase activities and greater NHase stability at 55„aC. The supplementation of these different sugars was shown to alter cellular and lipid bound trehalose levels, a sugar known to stabilize proteins and a component of the rhodococcal cell envelope. Cells that had higher levels of cellular trehalose had significantly greater NHase stability at 55„aC. The effect of the different sugar supplements and inducers of NHase, such as cobalt, on cell envelope components such as mycolic acids and glycolipids were examined by High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC). The results showed that changes in mycolic acids and glycolipids occurred when the cells were grown in the presence of different sugar supplements and when the cells were induced for NHase. Susceptibility of Rhodococcus sp DAP 96253 to different antibiotics was examined to indicate if changes were occurring in the cell envelope. Differences in antibiotic susceptibility were observed when the cells were grown on media with different sugar supplements and when the cells were induced for NHase. In the presence of cobalt Rhodococcus sp DAP 96253 showed a significant increase in sensitivity to antibiotics. Changes in growth media composition influences the cell envelope of Rhodococcus sp DAP 96253 and also affects NHase activity and stability. Therefore, achieving increased enzyme activity and stability is not entirely dependent on the actual enzyme, but is related to other aspects of the cell, such as the cell envelope and metabolites of the cell.

The bacterial cell wall, composed of peptidoglycan (PG), is an essential component of the cell envelope. This macromolecular structure fortifies the cell membrane, determines cell shape, and helps prevent osmotic lysis. The synthesis and remodeling/recycling of this polymer is mediated by PG synthases and hydrolases, respectively. Proper control of the PG hydrolases is particularly important since misregulation of these enzymes can lead to lethal breaches in the cell wall. Surprisingly, however, the precise molecular mechanisms governing the activities of these enzymes remain poorly understood. To help understand how PG hydrolases are regulated, I examined how their activity is controlled during cytokinesis in Escherichia coli. One important class of PG hydrolases necessary for cell division is the LytC-type amidases (AmiA, AmiB and AmiC). These enzymes require activation by the LytM factors EnvC and NlpD. My work focused on elucidating the mechanism by which the LytM factors activate the amidases. Using a genetic enrichment strategy, I isolated amiB misregulation mutants. Interestingly, the mutations mapped to a region of AmiB found only in cell separation amidases. Structural analysis of an AmiB ortholog indicates that this region corresponds to an alpha-helical domain that appears to occlude the active site. Thus, activation of the amidases by the LytM factors likely occurs via a conformational change that displaces the regulatory helix from the active site. In addition to amidase regulation, I also investigated how the LytM activators are recruited to, and regulated at the site of division. Using genetic and biochemical approaches, I showed that EnvC is directly recruited to the division site by FtsEX, an ATP-binding transporter- like complex. Interestingly, ATPase-defective FtsEX derivatives can still recruit EnvC to the divisome, but fail to promote cytokinesis. These results support a model where conformational changes induced by the ATPase activity of FtsE are directly and specifically transmitted to the amidases via FtsX and EnvC. This model is attractive because it provides a mechanism for converting the potentially dangerous activity of septal PG splitting into a discrete process which can be cycled on and off in coordination with the division process.

Šiame darbe tirtas gramteigiamųjų ir gramneigiamųjų bakterijų atsakas į natyvaus ir katalitiškai neaktyvaus lizocimo bei jo 9 aminorūgščių katijoninio peptido sukeltus ląstelės apvalkalo pažeidimus. Panaudojant potenciometrinius metodus, buvo nustatyta, kad šie junginiai sukelia viduląstelinio K+ ištekėjimą iš L. lactis, B. subtilis ir P. aeruginosa ląstelių ir dalinę jų citoplazminės membranos depoliarizaciją, tačiau natyvus, kaitintas lizocimas ir 9a peptidas skirtingai veikia L. lactis apvalkalėlio laidumą. Peptidas bei kaitintas lizocimas sukelia staigesnį K+ jonų ištekėjimą ir membranos įtampos sumažėjimą nei natyvus lizocimas. Peptido sukeliamas K+ jonų ištekėjimas yra grįžtamas. Taip pat buvo įvertintas įvairių mutantinių L. lactis padermių jautrumas lizocimui. Pastebėta, kad kuo padermė atsparesnė lizocimui, tuo vėliau prasideda K+ jonų ištekėjimas iš šios padermės ląstelių. Pasitelkus DNR mikrogardelių metodą, buvo tiriamas lizocimo bei peptido poveikis L. lactis MG1363 bei ΔoppA ląstelių genų raiškos pokyčiams. Nustatyta, jog peptidu ir lizocimu veiktose ląstelėse iššaukiama dvikomponentės valdymo sistemos CesSR raiška ir sukeliamas nuo SpxB priklausomas ląstelių atsparumas lizocimui. Gauti rezultatai rodo, jog lizocimas ir 9a peptidas sukelia kiek skirtingus L. lactis transkriptomo pokyčius: lizocimas savitai skatina nuo N-deacetilazės priklausomo atsparumo mechanizmo įsijungimą, taip pat skiriasi OpuA sistemą koduojančių genų raiška. / We used potenciometric measurments to investigate the response of Gram-positive and Gram-negative bacteria to cell envelope stress, caused by native and heat-inactivated lysozyme and lysozyme-derived 9 amino acid peptide. It was found that these antimicrobial compounds induce leakage of K+ outside the cells of L. lactis, B. subtilis and P. aeruginosa and cause partial depolarization of bacterial cytoplasmic membrane. We observed different response of L. lactis cells to these compounds – peptide and heat-inactivated lysozyme cause more rapid efflux of K+ ions than native lysozyme. Peptide has a reversible effect on K+ leakage. Sensitivity of different mutant strains of L. lactis to lysozyme was studied. It was shown that more resistant the strain is, the later the leakage of K+ ions is induced by lysozyme. To investigate the genome-wide response of L. lactis MG1363 and ΔoppA strains to lysozyme and 9 a.a. peptide, changes of gene expression after challenging cells with these antimicrobial compounds were analysed using DNA microarrays. It was estimated, that lysozyme and lysozyme-derived 9a.a. peptide induce CesSR system and SpxB-mediated response. It was also shown that L. lactis response to 9a.a. peptide and lysozyme differs. Lysozyme specifically induces PgdA-mediated resistance mechanism. Changes of expression of OpuA system in lysozyme and peptide treated cells are also different.

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