Requirement of NMB0065 for connecting assembly and export of sialic acid capsular polysaccharides in Neisseria meningitidis

Capsule expression in Neisseria meningitidis is encoded by the cps locus comprised of genes required for biosynthesis and surface translocation. Located adjacent to the gene encoding the polysialyltransferase in serogroups expressing sialic acid-containing capsule, NMB0065 is likely a member of the cps locus, but it is not found in serogroups A or X that express non-sialic acid capsules. To further understand its role in CPS expression, NMB0065 mutants were created in the serogroups B, C and Y strains. The mutants were as sensitive as unencapsulated strains to killing by normal human serum, despite producing near wild-type levels of CPS. Absence of surface expression of capsule was suggested by increased surface hydrophobicity and confirmed by immunogold electron microscopy, which revealed the presence of large vacuoles containing CPS within the cell. GC–MS and NMR analyses of purified capsule from the mutant revealed no apparent changes in polymer structures and lipid anchors. Mutants of NMB0065 homologues in other sialic acid CPS expressing meningococcal serogroups had similar phenotypes. Thus, NMB0065 (CtrG) is not involved in biosynthesis or lipidation of sialic acid-containing capsule but encodes a protein required for proper coupling of the assembly complex to the membrane transport complex allowing surface expression of CPS.     

Disruption of Escherichia coli Nissle 1917 K5 Capsule Biosynthesis,through Loss of Distinct kfi genes,Modulates Interaction with Intestinal Epithelial Cells and Impact on Cell Health

Escherichia coli Nissle 1917 (EcN) is among the best characterised probiotics, with a proven clinical impact in a range of conditions. Despite this, the mechanisms underlying these "probiotic effects" are not clearly defined. Here we applied random transposon mutagenesis to identify genes relevant to the interaction of EcN with intestinal epithelial cells. This demonstrated mutants disrupted in the kfiB gene, of the K5 capsule biosynthesis cluster, to be significantly enhanced in attachment to Caco-2 cells. However, this phenotype was distinct from that previously reported for EcN K5 deficient mutants (kfiC null mutants), prompting us to explore further the role of kfiB in EcN:Caco-2 interaction. Isogenic mutants with deletions in kfiB (EcNΔkfiB), or the more extensively characterised K5 capsule biosynthesis gene kfiC (EcNΔkfiC), were both shown to be capsule deficient, but displayed divergent phenotypes with regard to impact on Caco-2 cells. Compared with EcNΔkfiC and the EcN wild-type, EcNΔkfiB exhibited significantly greater attachment to Caco-2 cells, as well as apoptotic and cytotoxic effects. In contrast, EcNΔkfiC was comparable to the wild-type in these assays, but was shown to induce significantly greater COX-2 expression in Caco-2 cells. Distinct differences were also apparent in the pervading cell morphology and cellular aggregation between mutants. Overall, these observations reinforce the importance of the EcN K5 capsule in host-EcN interactions, but demonstrate that loss of distinct genes in the K5 pathway can modulate the impact of EcN on epithelial cell health.     

Genes Associated with Desiccation and Osmotic Stress in Listeria monocytogenes as Revealed by Insertional Mutagenesis

Listeria monocytogenes is a foodborne pathogen whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700 L. monocytogenes mutants was screened, using a microplate assay, for strains displaying increased or decreased desiccation survival (43% relative humidity, 15°C) in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food-grade stainless steel (SS) coupons in TSB plus 1% glucose (TSB-glu). Single transposon insertions in mutants exhibiting a change in desiccation survival of >0.5 log CFU/cm² relative to that of the wild type were determined by sequencing arbitrary PCR products. Strain morphology, motility, and osmotic stress survival (in TSB-glu plus 20% NaCl) were also analyzed. The initial screen selected 129 desiccation-sensitive (DS) and 61 desiccation-tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagellum-less strains contained transposons in genes involved in flagellum biosynthesis (fliP, flhB, flgD, flgL) and motor control (motB, fliM, fliY), while others harbored transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. The genes that were interrupted in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (P < 0.05) different survival relative to that of the wild type when exposed to osmotic stress, demonstrating that some genes likely have similar roles in allowing the organism to survive the two water stresses.     

Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by ‘mutant DNA fingerprints’

Whole genome sequencing of several microbes has revealed thousands of genes of unknown function. A large proportion of these genes seem to confer subtle quantitative phenotypes or phenotypes that do not have a plate screen. We report a novel method to monitor such phenotypes, where the fitness of mutants is assessed in mixed cultures under competitive growth conditions, and the abundance of any individual mutant in the pool is followed by means of its unique feature, namely the mutation itself. A mixed population of yeast mutants, obtained through transposon mutagenesis, was subjected to selection. The DNA regions (targets) flanking the transposon, until nearby restriction sites, are then quantitatively amplified by means of a ligation-mediated PCR method, using transposon-specific and adapter-specific primers. The amplified PCR products correspond to mutated regions of the genome and serve as ‘mutant DNA fingerprints’ that can be displayed on a sequencing gel. The relative intensity of the amplified DNA fragments before and after selection match with the relative abundance of corresponding mutants, thereby revealing the fate of the mutants during selection. Using this method we demonstrate that UBI4, YDJ1 and HSP26 are essential for stress tolerance of yeast during ethanol production. We anticipate that this method will be useful for functional analysis of genes of any microbe amenable to insertional mutagenesis.     

Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling

Klebsiella pneumoniae is an etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group has shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-κB canonical pathway. Our results revealed that K. pneumoniae capsule polysaccharide (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-of-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-κB. Two gene ontology categories accounted for half of the loci identified in the screening: metabolism and transport genes (32% of the mutants) and envelope-related genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression, which in turn underlined the NF-κB activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of the immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS O-polysaccharide and T2SS mutant-induced responses were dependent on TLR2-TLR4-MyD88 activation suggested that LPS O-polysaccharide and PulA perturbed Toll-like receptor (TLR)-dependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS O-polysaccharide and PulA T2SS could be new targets for the design of new antimicrobials. Increasing TLR-governed defense responses might provide also selective alternatives for the management of K. pneumoniae pneumonia.     

Type 1 fimbriae and extracellular polysaccharides are preeminent uropathogenic Escherichia coli virulence determinants in the murine urinary tract

Escherichia coli is the leading cause of urinary tract infections (UTIs). Despite the association of numerous bacterial factors with uropathogenic E. coli (UPEC), few such factors have been proved to be required for UTI in animal models. Previous investigations of urovirulence factors have relied on prior identification of phenotypic characteristics. We used signature-tagged mutagenesis (STM) in an unbiased effort to identify genes that are essential for UPEC survival within the murine urinary tract. A library of 2049 transposon mutants of the prototypic UPEC strain CFT073 was constructed using mini-Tn5km2 carrying 92 unique tags and screened in a murine model of ascending UTI. After initial screening followed by confirmation in co-infection experiments, 19 survival-defective mutants were identified. These mutants were recovered in numbers 101- to 106-fold less than the wild type in the bladder, kidneys or urine or at more than one site. The transposon junctions from each attenuated mutant were sequenced and analysed. Mutations were found in: (i) the type 1 fimbrial operon; (ii) genes involved in the biosyn-thesis of extracellular polysaccharides including group I capsule, group II capsule and enterobacterial common antigen; (iii) genes involved in metabolic pathways; and (iv) genes with unknown function. Five of the genes identified are absent from the genome of the E. coli K-12 strain. Mutations in type 1 fimbrial genes resulted in severely attenuated colonization, even in the case of a mutant with an insertion upstream of the fim operon that affected the rate of fimbrial switching from the 'off' to the 'on' phase. Three mutants had insertions in a new type II capsule biosynthesis locus on a pathogenicity island and were impaired in the production of capsule in vivo. An additional mutant with an insertion in wecE was unable to synthesize enterobacterial common antigen. These results confirm the pre-eminence of type 1 fimbriae, establish the importance of extracellular polysaccharides in the pathogenesis of UTI and identify new urovirulence determinants.     

Genetic Analysis and Prevalence Studies of the brp Exopolysaccharide Locus of Vibrio vulnificus

Phase variation in the Gram-negative human pathogen Vibrio vulnificus involves three colonial morphotypes- smooth opaque colonies due to production of capsular polysaccharide (CPS), smooth translucent colonies as the result of little or no CPS expression, and rugose colonies due to production of a separate extracellular polysaccharide (EPS), which greatly enhances biofilm formation. Previously, it was shown that the brp locus, which consists of nine genes arranged as an operon, is up-regulated in rugose strains in a c-di-GMP-dependent manner, and that plasmid insertions into the locus resulted in loss of rugosity and efficient biofilm production. Here, we have used non-polar mutagenesis to assess the involvement of individual brp genes in production of EPS and related phenotypes. Inactivation of genes predicted to be involved in various stages of EPS biosynthesis eliminated both the rugose colonial appearance and production of EPS, while knockout of a predicted flippase function involved in EPS transport resulted in a dry, lightly striated phenotype, which was associated with a reduction of brp-encoded EPS on the cell surface. All brp mutants retained the reduced motility characteristic of rugose strains. Lastly, we provide evidence that the brp locus is highly prevalent among strains of V. vulnificus.     

A Genetic Screen Using the PiggyBac Transposon in Haploid Cells Identifies Parp1 as a Mediator of Olaparib Toxicity

Genetic perturbation screens have the potential to dissect a wide range of cellular phenotypes. Such screens have historically been difficult in diploid mammalian cells. The recent derivation of haploid embryonic stem cells provides an opportunity to cause loss of function mutants with a random mutagen in a mammalian cell with a normal genetic background. We describe an approach to genetic screens that exploits the highly active piggyBac transposon in haploid mammalian cells. As an example of haploid transposon (HTP) screening, we apply this approach to identifying determinants of cancer drug toxicity and resistance. In a screen for 6-thioguanine resistance we recovered components of the DNA mismatch repair pathway, a known requirement for toxicity. In a further screen for resistance to the clinical poly(ADP-ribose) polymerase (PARP) inhibitor olaparib we recovered multiple Parp1 mutants. Our results show that olaparib toxicity to normal cells is mediated predominantly via Parp1, and suggest that the clinical side effects of olaparib may be on target. The transposon mutant libraries are stable and can be readily reused to screen other drugs. The screening protocol described has several advantages over other methods such as RNA interference: it is rapid and low cost, and mutations can be easily reverted to establish causality.     

Isolation and characterization of Francisella novicida mutants defective in lipopolysaccharide biosynthesis

In order to identify genes involved in LPS biosynthesis we isolated random mutants generated by transposon insertion in Francisella novicida. The resulting mutant bank yielded mutants with three distinct LPS phenotypes, and three representative mutants were chosen for further study. One mutant that had short O-antigen chains was sensitive to serum; this mutant and one other were more sensitive to killing by deoxycholate than control strains. The third mutant was resistant to deoxycholate killing but slightly sensitive to serum. The three mutants varied in their ability to grow in macrophages. The DNA sequences interrupted by the transposon in two of the three mutants showed similarity to known LPS biosynthetic genes at the deduced amino acid level.     

Transposon mutagenesis and cloning of the genes encoding the enzymes of fengycin biosynthesis inBacillus subtilis

A total of 20Bacillus subtilis F29-3 mutants defective in fengycin biosynthesis was obtained by Tn917 mutagenesis. Cloning and mapping results showed that the transposon in these mutants was inserted in eleven different locations on the chromosome. We were able to use the chromosomal sequence adjacent to the transposon as a probe to screen for cosmid clones containing the fengycin biosynthesis genes. One of the clones obtained, pFC660, was 46 kb long. Eight transposon insertion sites were mapped within this plasmid. Among the eleven different mutants analyzed, four mutants had Tn917 inserted in regions which encoded peptide sequences similar to part of gramicidin S synthetase, surfactin synthetase, and tyrocidine synthetase. Our results suggest that fengycin is synthesized nonribosomally by the multienzyme thiotemplate mechanism.     

Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'

Whole genome sequencing of several microbes has revealed thousands of genes of unknown function. A large proportion of these genes seem to confer subtle quantitative phenotypes or phenotypes that do not have a plate screen. We report a novel method to monitor such phenotypes, where the fitness of mutants is assessed in mixed cultures under competitive growth conditions, and the abundance of any individual mutant in the pool is followed by means of its unique feature, namely the mutation itself. A mixed population of yeast mutants, obtained through transposon mutagenesis, was subjected to selection. The DNA regions (targets) flanking the transposon, until nearby restriction sites, are then quantitatively amplified by means of a ligation-mediated PCR method, using transposon-specific and adapter-specific primers. The amplified PCR products correspond to mutated regions of the genome and serve as 'mutant DNA fingerprints' that can be displayed on a sequencing gel. The relative intensity of the amplified DNA fragments before and after selection match with the relative abundance of corresponding mutants, thereby revealing the fate of the mutants during selection. Using this method we demonstrate that UBI4, YDJ1 and HSP26 are essential for stress tolerance of yeast during ethanol production. We anticipate that this method will be useful for functional analysis of genes of any microbe amenable to insertional mutagenesis.     

Identification of Streptococcus agalactiae virulence genes in the neonatal rat sepsis model using signature-tagged mutagenesis

Group B streptococcal (GBS) infections are the most common cause of bacterial sepsis in the immediate newborn period. Apart from the capsule, the factors required for survival of GBS in the host are not well defined. In this study, signature-tagged transposon mutagenesis (STM) was used to identify genes required for growth and survival of GBS in a neonatal rat sepsis infection model. Approximately 1600 transposon mutants were screened in pools of 80 mutants, and approximately 120 mutants defective for survival in the animal host were identified. We successfully cloned and sequenced DNA flanking the transposon insertions from 92 of the mutants. Fifty per cent of the mutants had transposon insertions in genes with homologues in the public databases, whereas the remaining 50% had transposon insertions in genes with unknown function. A significant proportion of the avirulent mutants had transposon insertions in genes encoding transport-associated or regulatory proteins or in genes involved in cell surface metabolism, emphasizing the significance of these functions for in vivo survival of GBS. Overall, STM analysis revealed GBS genomic loci that encode a wide variety of functional gene classes, underscoring the diversity of bacterial processes required for the infection process. Currently, the function of the genes identified during the screening can only be inferred by homology to previously described genes. However, a number of the genes identified in this study have been shown to correlate with virulence in other pathogens. A virulence of a subset of mutants identified during the screening was confirmed by performing competitive index assays and lethal dose assays. This represents the first report of a genome-wide scan for virulence factors in GBS. The identified genes will further our understanding of the pathogenesis of GBS infections and may represent targets for intervention or lead to the development of novel therapies.     

Identification of genes involved in salt tolerance and symbiotic nitrogen fixation in chickpea rhizobium Mesorhizobium ciceri Ca181

In an attempt to find the genes involved in salt tolerance of the highly adaptable chickpea rhizobium strain, Mesorhizobium ciceri Ca181, a Tn5 transposon insertion library was generated and screened to identify five mutants with inability to survive in the presence of 0.1 M NaCl. The genes disrupted in these mutants due to insertion of the transposon were identified by sequencing of Tn5 flanking sequences after inverse PCR. One of the mutants had a disruption in diguanylate cyclase gene which is involved in bacterial biofilm formation and persistence. The second mutant had a disruption in an ABC transporter membrane protein gene, which is involved in the uptake of nutrients and cellular osmoprotection. The third mutant had a disruption in a gene showing homology with rhamnulose 1-phosphate aldolase which has an important role in the central metabolism of L-rhamnulose. The fourth mutant had a disruption in a capsule synthesis gene and the fifth mutant had an insertion in an oxidoreductase gene. When these mutants were inoculated into the host chickpea plant under normal non-saline conditions, they formed symbiotic nodules but with severely reduced nitrogenase activity. Hence, it appears that bacterial ability to adapt to hyper-osmotic salt stress conditions is also important for its nitrogen fixing ability in the chickpea root nodules. Allele mining for variant forms of the identified genes in the germplasm resources of M. ciceri may help in the development of highly adaptive and efficient nitrogen fixing strains of the chickpea rhizobium.     

Phase-Variable Surface Structures Are Required for Infection of Campylobacter jejuni by Bacteriophages

This study characterizes the interaction between Campylobacter jejuni and the 16 phages used in the United Kingdom typing scheme by screening spontaneous mutants of the phage-type strains and transposon mutants of the sequenced strain NCTC 11168. We show that the 16 typing phages fall into four groups based on their patterns of activity against spontaneous mutants. Screens of transposon and defined mutants indicate that the phage-bacterium interaction for one of these groups appears to involve the capsular polysaccharide (CPS), while two of the other three groups consist of flagellatropic phages. The expression of CPS and flagella is potentially phase variable in C. jejuni, and the implications of these findings for typing and intervention strategies are discussed.     

Autophosphorylation of the Bacterial Tyrosine-Kinase CpsD Connects Capsule Synthesis with the Cell Cycle in Streptococcus pneumoniae

Bacterial capsular polysaccharides (CPS) are produced by a multi-protein membrane complex, in which a particular type of tyrosine-autokinases named BY-kinases, regulate their polymerization and export. However, our understanding of the role of BY-kinases in these processes remains incomplete. In the human pathogen Streptococcus pneumoniae, the BY-kinase CpsD localizes at the division site and participates in the proper assembly of the capsule. In this study, we show that the cytoplasmic C-terminal end of the transmembrane protein CpsC is required for CpsD autophosphorylation and localization at mid-cell. Importantly, we demonstrate that the CpsC/CpsD complex captures the polysaccharide polymerase CpsH at the division site. Together with the finding that capsule is not produced at the division site in cpsD and cpsC mutants, these data show that CPS production occurs exclusively at mid-cell and is tightly dependent on CpsD interaction with CpsC. Next, we have analyzed the impact of CpsD phosphorylation on CPS production. We show that dephosphorylation of CpsD induces defective capsule production at the septum together with aberrant cell elongation and nucleoid defects. We observe that the cell division protein FtsZ assembles and localizes properly although cell constriction is impaired. DAPI staining together with localization of the histone-like protein HlpA further show that chromosome replication and/or segregation is defective suggesting that CpsD autophosphorylation interferes with these processes thus resulting in cell constriction defects and cell elongation. We show that CpsD shares structural homology with ParA-like ATPases and that it interacts with the chromosome partitioning protein ParB. Total internal reflection fluorescence microscopy imaging demonstrates that CpsD phosphorylation modulates the mobility of ParB. These data support a model in which phosphorylation of CpsD acts as a signaling system coordinating CPS synthesis with chromosome segregation to ensure that daughter cells are properly wrapped in CPS.     

Tyrosine phosphorylation of CpsD negatively regulates capsular polysaccharide biosynthesis in streptococcus pneumoniae

In Streptococcus pneumoniae, the first four genes of the capsule locus (cpsA to cpsD) are common to most serotypes. By analysis of various in-frame deletion and site-directed mutants, the function of their gene products in capsular polysaccharide (CPS) biosynthesis was investigated. We found that while CpsB, C and D are essential for encapsulation, CpsA is not. CpsC and CpsD have similarity to the amino-terminal and carboxy-terminal regions, respectively, of the autophosphorylating protein-tyrosine kinase Wzc from Escherichia coli. Alignment of CpsD with Wzc and other related proteins identified conserved Walker A and B sequence motifs and a tyrosine rich domain close to the carboxy-terminus. We have shown that CpsD is also an autophosphorylating protein-tyrosine kinase and that point mutations in cpsD affecting either the ATP-binding domain (Walker A motif) or the carboxy-terminal [YGX]4 repeat domain eliminated tyrosine phosphorylation of CpsD. We describe, for the first time, the phenotypic impact of these two mutations on polysaccharide production and show that they affect CPS production differently. Whereas a mutation in the Walker A motif resulted in loss of encapsulation, mutation of the tyrosines in the [YGX]4 repeat domain resulted in an apparent increase in encapsulation and a mucoid phenotype. These data suggest that autophosphorylation of CpsD at tyrosine attenuates its activity and reduces the level of encapsulation. Additionally, we demonstrated that CpsC is required for CpsD tyrosine phosphorylation and that CpsB influences dephosphorylation of CpsD. These results are consistent with CpsD tyrosine phosphorylation acting to negatively regulate CPS production. This has implications for the function of CpsC/CpsD homologues in both Gram-positive and Gram-negative bacteria and provides a mechanism to explain regulation of CPS production during pathogenesis.