The role of galacturonic acid in outer membrane stability in Klebsiella pneumoniae.

In most members of the Enterobacteriaceae, including Escherichia coli and Salmonella, the lipopolysaccharide core oligosaccharide backbone is modified by phosphoryl groups. The negative charges provided by these residues are important in maintaining the barrier function of the outer membrane. Mutants lacking the core heptose region and the phosphate residues display pleiotrophic defects collectively known as the deep-rough phenotype, characterized by changes in outer membrane structure and function. Klebsiella pneumoniae lacks phosphoryl residues in its core, but instead contains galacturonic acid. The goal of this study was to determine the contribution of galacturonic acid as a critical source of negative charge. A mutant was created lacking all galacturonic acid by targeting UDP-galacturonic acid precursor synthesis through a mutation in gla(KP). Gla(KP) is a K. pneumoniae UDP-galacturonic acid C4 epimerase providing UDP-galacturonic acid for core synthesis. The gla(KP) gene was inactivated and the structure of the mutant lipopolysaccharide was determined by mass spectrometry. The mutant displayed characteristics of a deep-rough phenotype, exhibiting a hypersensitivity to hydrophobic compounds and polymyxin B, an altered outer membrane profile, and the release of the periplasmic enzyme beta-lactamase. These results indicate that the negative charge provided by the carboxyl groups of galacturonic acid do play an equivalent role to the core oligosaccharide phosphate residues in establishing outer membrane integrity in E. coli and Salmonella

Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis

Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.

Towards an automated analysis of bacterial peptidoglycan structure.

Peptidoglycan (PG) is an essential component of the bacterial cell envelope. This macromolecule consists of glycan chains alternating N-acetylglucosamine and N-acetylmuramic acid, cross-linked by short peptides containing nonstandard amino acids. Structural analysis of PG usually involves enzymatic digestion of glycan strands and separation of disaccharide peptides by reversed-phase HPLC followed by collection of individual peaks for MALDI-TOF and/or tandem mass spectrometry. Here, we report a novel strategy using shotgun proteomics techniques for a systematic and unbiased structural analysis of PG using high-resolution mass spectrometry and automated analysis of HCD and ETD fragmentation spectra with the Byonic software. Using the PG of the nosocomial pathogen Clostridium difficile as a proof of concept, we show that this high-throughput approach allows the identification of all PG monomers and dimers previously described, leaving only disambiguation of 3-3 and 4-3 cross-linking as a manual step. Our analysis confirms previous findings that C. difficile peptidoglycans include mainly deacetylated N-acetylglucosamine residues and 3-3 cross-links. The analysis also revealed a number of low abundance muropeptides with peptide sequences not previously reported. Graphical Abstract The bacterial cell envelope includes plasma membrane, peptidoglycan, and surface layer. Peptidoglycan is unique to bacteria and the target of the most important antibiotics; here it is analyzed by mass spectrometry

PCR-Based Identification of Klebsiella pneumoniae subsp. rhinoscleromatis, the Agent of Rhinoscleroma

Rhinoscleroma is a chronic granulomatous infection of the upper airways caused by the bacterium Klebsiella pneumoniae subsp. rhinoscleromatis. The disease is endemic in tropical and subtropical areas, but its diagnosis remains difficult. As a consequence, and despite available antibiotherapy, some patients evolve advanced stages that can lead to disfiguration, severe respiratory impairment and death by anoxia. Because identification of the etiologic agent is crucial for the definitive diagnosis of the disease, the aim of this study was to develop two simple PCR assays. We took advantage of the fact that all Klebsiella pneumoniae subsp. rhinoscleromatis isolates are (i) of capsular serotype K3; and (ii) belong to a single clone with diagnostic single nucleotide polymorphisms (SNP). The complete sequence of the genomic region comprising the capsular polysaccharide synthesis (cps) gene cluster was determined. Putative functions of the 21 genes identified were consistent with the structure of the K3 antigen. The K3-specific sequence of gene Kr11509 (wzy) was exploited to set up a PCR test, which was positive for 40 K3 strains but negative when assayed on the 76 other Klebsiella capsular types. Further, to discriminate Klebsiella pneumoniae subsp. rhinoscleromatis from other K3 Klebsiella strains, a specific PCR assay was developed based on diagnostic SNPs in the phosphate porin gene phoE. This work provides rapid and simple molecular tools to confirm the diagnostic of rhinoscleroma, which should improve patient care as well as knowledge on the prevalence and epidemiology of rhinoscleroma

Independent evolution of shape and motility allows evolutionary flexibility in Firmicutes bacteria

Functional morphological adaptation is an implicit assumption across many ecological studies. However, despite a few pioneering attempts to link bacterial form and function, functional morphology is largely unstudied in prokaryotes. One intriguing candidate for analysis is bacterial shape, as multiple lines of theory indicate that cell shape and motility should be strongly correlated. Here we present a large-scale use of modern phylogenetic comparative methods to explore this relationship across 325 species of the phylum Firmicutes. In contrast to clear predictions from theory, we show that cell shape and motility are not coupled, and that transitions to and from flagellar motility are common and strongly associated with lifestyle (free-living or host-associated). We find no association between shape and lifestyle, and contrary to recent evidence, no indication that shape is associated with pathogenicity. Our results suggest that the independent evolution of shape and motility in this group might allow a greater evolutionary flexibility

Genetic Dissection of an Exogenously Induced Biofilm in Laboratory and Clinical Isolates of E. coli

Microbial biofilms are a dominant feature of many human infections. However, developing effective strategies for controlling biofilms requires an understanding of the underlying biology well beyond what currently exists. Using a novel strategy, we have induced formation of a robust biofilm in Escherichia coli by utilizing an exogenous source of poly-N-acetylglucosamine (PNAG) polymer, a major virulence factor of many pathogens. Through microarray profiling of competitive selections, carried out in both transposon insertion and over-expression libraries, we have revealed the genetic basis of PNAG-based biofilm formation. Our observations reveal the dominance of electrostatic interactions between PNAG and surface structures such as lipopolysaccharides. We show that regulatory modulation of these surface structures has significant impact on biofilm formation behavior of the cell. Furthermore, the majority of clinical isolates which produced PNAG also showed the capacity to respond to the exogenously produced version of the polymer

Multiple Peptidoglycan Modification Networks Modulate Helicobacter pylori's Cell Shape, Motility, and Colonization Potential

Helical cell shape of the gastric pathogen Helicobacter pylori has been suggested to promote virulence through viscosity-dependent enhancement of swimming velocity. However, H. pylori csd1 mutants, which are curved but lack helical twist, show normal velocity in viscous polymer solutions and the reason for their deficiency in stomach colonization has remained unclear. Characterization of new rod shaped mutants identified Csd4, a DL-carboxypeptidase of peptidoglycan (PG) tripeptide monomers and Csd5, a putative scaffolding protein. Morphological and biochemical studies indicated Csd4 tripeptide cleavage and Csd1 crosslinking relaxation modify the PG sacculus through independent networks that coordinately generate helical shape. csd4 mutants show attenuation of stomach colonization, but no change in proinflammatory cytokine induction, despite four-fold higher levels of Nod1-agonist tripeptides in the PG sacculus. Motility analysis of similarly shaped mutants bearing distinct alterations in PG modifications revealed deficits associated with shape, but only in gel-like media and not viscous solutions. As gastric mucus displays viscoelastic gel-like properties, our results suggest enhanced penetration of the mucus barrier underlies the fitness advantage conferred by H. pylori's characteristic shape

Quantitative Analyses Reveal Novel Roles for N-Glycosylation in a Major Enteric Bacterial Pathogen

In eukaryotes, glycosylation plays a role in proteome stability, protein quality control, and modulating protein function; however, similar studies in bacteria are lacking. Here, we investigate the roles of general protein glycosylation systems in bacteria using the enteropathogen Campylobacter jejuni as a well-defined example. By using a quantitative proteomic strategy, we were able to monitor changes in the C. jejuni proteome when glycosylation is disrupted. We demonstrate that in C. jejuni , N-glycosylation is essential to maintain proteome stability and protein quality control. These findings guided us to investigate the role of N-glycosylation in modulating bacterial cellular activities. In glycosylation-deficient C. jejuni, the multidrug efflux pump and electron transport pathways were significantly impaired. We demonstrate that in vivo, fully glycosylation-deficient C. jejuni bacteria were unable to colonize its natural avian host. These results provide the first evidence of a link between proteome stability and complex functions via a bacterial general glycosylation system.IMPORTANCE Advances in genomics and mass spectrometry have revealed several types of glycosylation systems in bacteria. However, why bacterial proteins are modified remains poorly defined. Here, we investigated the role of general N-linked glycosylation in a major food poisoning bacterium, Campylobacter jejuni The aim of this study is to delineate the direct and indirect effects caused by disrupting this posttranslational modification. To achieve this, we employed a quantitative proteomic strategy to monitor alterations in the C. jejuni proteome. Our quantitative proteomic results linked general protein N-glycosylation to maintaining proteome stability. Functional analyses revealed novel roles for bacterial N-glycosylation in modulating multidrug efflux pump, enhancing nitrate reduction activity, and promoting host-microbe interaction. This work provides insights on the importance of general glycosylation in proteins in maintaining bacterial physiology, thus expanding our knowledge of the emergence of posttranslational modification in bacteria

Evasion of IFN-γ Signaling by Francisella novicida Is Dependent upon Francisella Outer Membrane Protein C

Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of the lethal disease tularemia. An outer membrane protein (FTT0918) of F. tularensis subsp. tularensis has been identified as a virulence factor. We generated a F. novicida (F. tularensis subsp. novicida) FTN_0444 (homolog of FTT0918) fopC mutant to study the virulence-associated mechanism(s) of FTT0918.The ΔfopC strain phenotype was characterized using immunological and biochemical assays. Attenuated virulence via the pulmonary route in wildtype C57BL/6 and BALB/c mice, as well as in knockout (KO) mice, including MHC I, MHC II, and µmT (B cell deficient), but not in IFN-γ or IFN-γR KO mice was observed. Primary bone marrow derived macrophages (BMDM) prepared from C57BL/6 mice treated with rIFN-γ exhibited greater inhibition of intracellular ΔfopC than wildtype U112 strain replication; whereas, IFN-γR KO macrophages showed no IFN-γ-dependent inhibition of ΔfopC replication. Moreover, phosphorylation of STAT1 was downregulated by the wildtype strain, but not the fopC mutant, in rIFN-γ treated macrophages. Addition of NG-monomethyl-L-arginine, an NOS inhibitor, led to an increase of ΔfopC replication to that seen in the BMDM unstimulated with rIFN-γ. Enzymatic screening of ΔfopC revealed aberrant acid phosphatase activity and localization. Furthermore, a greater abundance of different proteins in the culture supernatants of ΔfopC than that in the wildtype U112 strain was observed.F. novicida FopC protein facilitates evasion of IFN-γ-mediated immune defense(s) by down-regulation of STAT1 phosphorylation and nitric oxide production, thereby promoting virulence. Additionally, the FopC protein also may play a role in maintaining outer membrane stability (integrity) facilitating the activity and localization of acid phosphatases and other F. novicida cell components