The ∆nfs and exo mutants display similar phenotypes both in starvation- and glycerol-induced sporulation. To test whether the gene products function in the same process during spore formation, we first examined whether both mutants share the same dependency on developmental marker genes during starvation-induced sporulation. To be able to quantify the activity of the nfs promoter in various developmental mutant backgrounds, we fused the putative nfs promoter region to mcherry as a reporter.
The quantitative fluorescence measurements of developing cultures suggest that nfs expression during starvation-induced development is entirely dependent on csgA and therefore on C-signaling. The csgA-mutant is blocked early in development; it does not aggregate or sporulate. In the dev mutant background however, nfs promoter-driven gene expression was detected, but less than in the wild type. The dev-mutant does not form mature fruiting bodies and the number of spores is strongly reduced (Boysen et al., 2002). This result suggests that nfs-expression depends partially on the dev locus. In the exo mutant background, wild type levels of nfs expression were detected suggesting that nfs regulation is independent of exo.
fruA is also a non-developing mutant. The FruA-protein is a transcription factor with a central role during starvation-induced development. The protein is thought to be phosphorylated and this modified form is proposed to allow the developmental program to proceed by differential control of gene expression (Figure 1-6) (Ellehauge et al., 1998).
Unexpectedly, we saw a strong and early increase of nfs promoter-driven fluorescence in the fruA mutant compared to wild type. This result was confirmed by Nfs immunoblot on time-course samples form an independent fruA mutant strain (data not shown). Interestingly, unpublished results (X. Shi & L. Søgaard-Andersen) suggest that exo is likewise transcribed in the non-developing fruA mutant. A possible explanation is that nfs as well as exo-expression during starvation-induced development is controlled by FruA and that FruA is involved in repression of these genes during early development (Figure 3-1).
fruA
FruA FruA~P devRST DevT
nfs nfsCsgA
Early development Late development
Figure 3-1 Schematic model of nfs expression control. During early stages of development, unphosphorylated FruA represses nfs expression directly or indirectly. CsgA triggers FruA phosphorylation. Phosphorylated FruA induces devT expression and DevT stimulates fruA-expression. High levels of FruA~P relive repression of late developmental genes such as nfs.
However, fluorescence measurements cannot provide direct evidence for accumulation of the Nfs-proteins in spore forming cells. Accumulation of the signal does not necessarily reflect increasing promoter activity since the Mcherry protein may have different stability properties than the Nfs-proteins. An important next step to investigate whether nfs and exo participate in the same process is to generate a ∆nfs exo double mutant. Analysis of glycerol- and starvation- induced ∆nfs, exo and ∆nfs exo mutants as well as wild type by electron microscopy should reveal if there are similar defects or missing layers in the spheres.
3.3.1 The Nfs proteins likely participate in a cell envelope-associated functional complex
The observation that the single nfs in-frame deletion mutants display phenotypes similar to deletion of the entire locus suggests that the proteins function together. This assumption is supported by the reduced stability of the Nfs proteins in single gene deletion backgrounds.
The Nfs proteins are detectable by immunoblot 30 minutes after addition of glycerol. At this time, gene expression levels are already at maximum. After two hours, all six detectable proteins accumulate to highest levels but only NfsA and B are stably detected until twelve hours. Analysis of protein stabilities in the single gene deletion backgrounds have shown that stability of NfsD to H strongly depends on presence of NsfA, B and C. However, if NfsD to H are missing, NfsA, B, to C levels are only reduced. These results could mean that the Nfs proteins do not form one large tight complex but two interacting complexes consisting of NfsA, B, C and of NfsD to H.
Alternatively, NfsA, B and C may constitute a specific part of the complex that localizes to a different compartment (such as the outer membrane) and is therefore more stable (i.e. protected from degradation) than the remaining Nfs proteins. This possibility is supported by CELLO localization predictions that suggest that the first three proteins localize to the outer membrane.
NfsA and H likely are beta-barrel fold containing proteins. Beta-barrel structures are a characteristic motif of outer membrane proteins. NfsD contains a predicted N-terminal transmembrane domain. Therefore, the protein probably localizes to the cytoplasmic membrane. For NfsG, psiBlastp alignments suggest that this protein is related to ABC-transporters or ATP binding proteins. Additionally, Phobius prediction suggests that the N-terminal part is cytoplasm-exposed (containing the FHA-domain) and the C-terminal part localizes to the periplasm. Both parts are separated by a hydrophobic segment.
Therefore, the protein likely also localizes to the cytoplasmic membrane. NfsE contains a lipid attachment site. Based on the amino acid sequence (+2 D sorting rule, (Seydel et al., 1999)), the protein likely localizes to the outer membrane. For NfsB, C and F no clear prediction for a membrane anchoring structure or modification exists. Detection of NfsB and C in the cell envelope fraction by immunoblot suggests that those proteins are in a complex with the membrane anchored Nfs-proteins. NfsB and C share the protein stability pattern with NfsA suggesting that those proteins are in an outer membrane associated complex. NfsF contains a predicted signal peptide but no structural motif.
The protein was not detectable by immunoblot. Therefore, localization and putative interaction partners for the small protein are not certain.
Based on the available data, we propose the following organization of the Nfs-proteins in the cell envelope (Figure 3-2):
NfsA
NfsB NfsC
NfsF NfsE TPR
NfsG TPR ?
FHA
NfsH
NfsD
OM
IM
Figure 3-2 Schematic model of subcellular Nfs protein localization. OM: Outer membrane, IM: Inner membrane.
The predicted association of NfsA, B, C, E and H with the outer membrane and NfsD and G with the inner membrane needs to be confirmed by immunoblot of each separated membrane. To prove the assumed complex formation of the Nfs-Proteins, cross-linking and co-immunoprecipitation can be applied. To determine the stoichiometry of the proteins in the putative complex, quantitative immunoblot or in vitro complex formation analyses need to be performed. In vivo interaction can indirectly be shown by co-localization (fluorescent labelling or immunofluorescence) and directly by FRET-experiments with fluorescent markers that are known to function in the cell envelope such as Mcherry or Gfp and its derivatives when exported by the Tat-system.
During starvation-induced development, only two Nfs proteins were detected due to the strongly decreasing protein content of a developing culture and the fact that only a small proportion of cells convert into spores. To confirm that the Nfs proteins accumulate similarly during starvation-induced sporulation, the immunoblot analyses have to be carried out on samples of the spore-forming subpopulation. Recently, the experimental conditions for separation of cell subpopulations have been determined (B. Lee & P.
Higgs, unpublished). This approach should improve immunoblot results since it specifically enriches nfs expressing cells (only spore forming cells activate the nfs promoter as shown by fluorescence microscopy).
The reason for the inability to delete nfsC is intriguing. The protein contains a predicted domain of unknown function and may be related to prenylyltransferases. It is unlikely that the protein is essential since the entire locus can be deleted without apparent influence on vegetative growth. The inabilities to even recombine in either the up- or downstream region of the gene may suggest that this particular genomic region is difficult to access by DNA-recombination approaches.
3.3.2 The Nfs-proteins are probably involved in cell envelope modifications
Position specific iterative Blastp searches suggested that the Nsf-proteins could be related to glycosyl- and glucosamintransferases. One interesting result of the array analysis supporting the psiBlastp results is that the four mutants fdgA, Ω7536 (exo), Mxan_1101 and Mxan_3026 defective in glycerol- and, in case of exo and fdgA also starvation-induced spore formation, contain mutations in genes belonging to the GO-functional sub-category ‘Biosynthesis and degradation of surface polysaccharides and lipopolysaccharides’. Five of the ten up-regulated genes in this category are annotated as glycosyltransferases or N-acetylglucosamine epimerases which matches the psiBlastp results for NfsC, D and E. Glycosyltransferases form a large family of enzymes that catalyze the transfer of sugars or aminosugars to various substrates including proteins, lipids and peptidoglycan (Lairson et al., 2008) (Mohammadi et al., 2007). Therefore, putative functions of the Nfs proteins could be glycosylation of spore-specific proteins or their target could be the cell wall, i.e. they could be involved in coordinated cell wall modification or degradation during spore formation. If the peptidoglycan sacculus is not properly disassembled during spore formation but, for example, stays present either completely or as patches of cell envelope associated murein, spore formation could be perturbed. Interestingly, in E. coli, aberrant branched and spiral-like cell shapes have been reported to be associated with patches of inert peptidoglycan (de Pedro et al., 2003) and inhibition of penicillin binding proteins (Varma & Young, 2004). To analyze if the ∆nfs mutant is defective in these putative functions, both protein glycosylation and peptidoglycan synthesis of the ∆nfs mutant were investigated.
Most studies about protein glycosylation have been carried out in eukaryotic systems.
Here, glycosylation is one of the most important post-translational protein modifications. However, there exist an increasing number of glycosylated bacterial proteins. Interestingly, in bacteria surface exposed proteins are frequently glycosylated such as pilins, adhesins, S-layer proteins (Ku et al., 2009, Voisin et al., 2005, Steiner et al., 2007, Messner et al., 2008) as well as cortex and exosporium proteins of Bacillus
spores. Apparently, glycosylation of surface proteins utilizes different modules of the biosynthesis routes of lipopolysaccharide O-antigens. The sugar moieties are transferred from an activated precursor either as mono- or as oligosaccharide to the target protein by glycosyltransferases. In E. coli, this reaction has been shown to take place in the periplasm. Interestingly, in S. parasanguinis, a cluster of seven genes has recently been identified involved in biogenesis and glycosylation of a conserved surface glycoprotein and interaction of glycosyltransferases has also been shown (Bu et al., 2008) coinciding with the assumed interaction of the Nfs proteins. In M. xanthus, at least one surface glycoprotein (VGP) has been described (Glufka & Maeba, 1991) suggesting that M.
xanthus possesses the ability to modify exported proteins by glycosylation. Therefore, we reasoned that one possible function of the Nfs-proteins could be to glycosylate and facilitate export of spore coat associated target proteins.
Since all detectable Nfs-proteins migrate approximately at their calculated molecular mass in SDS-PA-gels, they are probably not extensively modified themselves by glycosylation. A hypothetical target protein is therefore likely not part of the nfs locus.
However, clear differences in protein glycosylation patterns between wild type and ∆nfs were not detectable with the applied staining method. Techniques with better resolution such as chromatography and mass spectrometry could provide clearer results and samples of cells that have been induced for more than four hours should also be included because spore coat proteins may be synthesized and exported very late.
Additionally, separated spore coats or outer membrane fractions of induced cells could be used for these analyses to enrich for the putative target protein(s).
Another cell envelope structure that is extensively modified both during spore formation and germination is the murein sacculus. In particular, upon spore germination, the peptidoglycan cell wall needs to be re-synthesized. To check if there are differences in cell wall synthesis during vegetative growth and germination in wild type and the ∆nfs mutant, staining of nascent peptidoglycan with Fluorescin labeled Vancomycin (VanFL) was applied. As expected, differences during vegetative growth were not observed consistent with the assumption that the Nfs proteins function specifically during spore formation. However, preparation of samples from outgrowing cells proved difficult because cells in this stage were very fragile. Therefore, modified fixing and staining methods should be applied. For instance, cells could be fixed on poly-L-lysine coated slides or treated on micropore filters to avoid mechanical forces. Nevertheless, some germinating cells displayed bright fluorescence signals at the tips of outgrowing spheres or patches of labeled peptidoglycan as expected suggesting that an optimized procedure could help to understand how outgrowing spores synthesize peptidoglycan de novo and organize it into a rod-shaped sacculus.
An additional possibility is that the Nfs proteins could be directly involved in synthesis of the carbohydrate containing spore envelope. Biogenesis and assembly of outer membrane components such as lipid A as well as the O-antigen carbohydrate chains and exopolysaccharides is accomplished by export from the inner leaflet of the cytoplasmic membrane, transport through the periplasm and insertion in the outer membrane. This process can involve a type I-like secretion system (Bos et al., 2007). Type I secretion systems consist of an inner-membrane ABC-transporter, an outer membrane protein and a ‘membrane fusion protein’ which connects the inner- and outer membrane
components (Gentschev et al., 2002, Gentschev et al., 2004) (Faridmoayer et al., 2007).
The M. xanthus spore carbohydrate translocation and exporting machinery could be similarly organized and the cell envelope associated Nfs proteins may be part of this machinery. NfsG is a good candidate for an inner membrane transporter since it probably localizes to the cytoplasmic membrane, is related to ABC-transporters and possesses a cytoplasmic FHA domain that could mediate interaction with a precursor-delivering protein or nucleotide-activated sugar moiety. NfsA and H could represent the outer membrane part of the functional entity and the other Nfs proteins could mediate delivery of the carbohydrates through the periplasmic space.
It is still unknown whether the carbohydrates in the spore coat are cross-linked. If they are, the cross-linking enzymes need to localize in the cell envelope. A possible task of the Nfs proteins could therefore also be to crosslink the subunits of the spore coat. If the carbohydrates are synthesized and exported but not cross-linked, the subunits (i.e.
glucose and galactosamine) may be found in the medium.
Additional evidence about the function of the Nfs proteins could also be obtained from an envelope analysis of the ovoids formed by the ∆nfs and exo mutants during glycerol induction and the spore-like entities formed inside fruiting bodies by electron microscopy. Furthermore, differences in the carbohydrate contents between ∆nfs and exo as well as wild type spores could help to reveal what the M. xanthus spore envelope consists of. Additionally, the phenotype of an nfs constitutively or overexpressing mutant could provide insights in the mechanism of how the Nfs proteins function.
Therefore, the strong pilA promoter could be cloned in front of the nfs-locus. Providing this mutant is viable, an aberrant envelope of vegetative cells and cell shape defects are possible.
3.4 The fate of the rod-shape determining protein MreB