Srishti Gupta

Identification of New Proteins that Modulate the Germination of Spores of Bacillus Species

A number of operons encoding the nutrient germinant receptors (GRs) in dormant spores of Bacillus megaterium and Bacillus subtilis species have small open reading frames (ORFs) of unknown function within or immediately adjacent to the operons. Inactivation of the genes in these ORFs, encoding proteins now termed D proteins, either significantly increased or decreased spore germination via the associated GR but had no effects on germination via non-GR-dependent germinants. These effects on GR-dependent germination were complemented by ectopic expression of the appropriate D gene (gene encoding D protein). However, substitution of noncognate D genes in two GR operons resulted in inhibition of germination via the GR manipulated, although ectopic overexpression of a D gene had no effect on overall GR-dependent germination. The various D genes studied were expressed in the forespore during sporulation in parallel with the associated GR operon, and transcription of a B. subtilis D gene was controlled by RNA polymerase sigma factor σ(G). These results indicate that proteins encoded by small ORFs within or adjacent to operons encoding GRs play major roles in modulating GR function in spores of Bacillus species. In B. subtilis, deletion of a D gene (B. subtilis gerKD [gerKDbs]) adjacent to the gerK operon encoding the GerK GR or ectopic expression or overexpression of gerKDbs had no major effect on the levels of GR subunits or of two other germination proteins.

Investigating the Functional Hierarchy of Bacillus megaterium PV361 Spore Germinant Receptors

Spores of Bacillus megaterium QM B1551 germinate rapidly when exposed to a number of single-trigger germinant compounds, including glucose, proline, leucine, and certain inorganic salts. However, spores of strain PV361, a plasmidless QM B1551 derivative that lacks the GerU germinant receptor (GR) responsible for mediating germination in response to single-trigger compounds, can germinate efficiently when incubated in nutritionally rich media, presumably via activation of additional germinant receptors. In this work, we have identified five chromosomally encoded GRs and attempted to characterize, by mutational analysis, germinant recognition profiles associated with the respective receptors in strain PV361. Of strains engineered with single GR insertion-deletions, only GerK-null spores displayed significant defective germination phenotypes when incubated in 5% (wt/vol) beef extract or plated on rich solid medium. Cumulative decreases in viability were observed in GerK-null spores that also lacked GerA or GerA2, indicating that these GRs, which exerted little effect on spore germination when disrupted individually, have a degree of functionality. Unexpectedly, an efficient germination response to combinations of germinants was restored in GerA(+) spores, which lack all other functional GRs, providing evidence for negative cooperativity between some GRs within the spore. Tetrazolium-based germinative assays conducted with purified spores indicated that these newly characterized B. megaterium GRs are cognate for a wide and chemically diverse range of germinant molecules, but unlike GerU, can only be induced to trigger germination when stimulated by at least two different germinants.

Motility defects in Campylobacter jejuni defined gene deletion mutants caused by second-site mutations

Genetic variation due to mutation and phase variation has a considerable impact on the commensal and pathogenic behaviours of Campylobacter jejuni. In this study, we provide an example of how second-site mutations can interfere with gene function analysis in C. jejuni. Deletion of the flagellin B gene (flaB) in C. jejuni M1 resulted in mutant clones with inconsistent motility phenotypes. From the flaB mutant clones picked for further analysis, two were motile, one showed intermediate motility and two displayed severely attenuated motility. To determine the molecular basis of this differential motility, a genome resequencing approach was used. Second-site mutations were identified in the severely attenuated and intermediate motility flaB mutant clones: a TA-dinucleotide deletion in fliW and an A deletion in flgD, respectively. Restoration of WT fliW, using a newly developed genetic complementation system, confirmed that the second-site fliW mutation caused the motility defect as opposed to the primary deletion of flaB. This study highlights the importance of (i) screening multiple defined gene deletion mutant clones, (ii) genetic complementation of the gene deletion and ideally (iii) screening for second-site mutations that might interfere with the pathways/mechanisms under study.

Genome-wide fitness analyses of the foodborne pathogen Campylobacter jejuni in in vitro and in vivo models

Campylobacter is the most common cause of foodborne bacterial illness worldwide. Faecal contamination of meat, especially chicken, during processing represents a key route of transmission to humans. There is a lack of insight into the mechanisms driving C. jejuni growth and survival within hosts and the environment. Here, we report a detailed analysis of C. jejuni fitness across models reflecting stages in its life cycle. Transposon (Tn) gene-inactivation libraries were generated in three C. jejuni strains and the impact on fitness during chicken colonisation, survival in houseflies and under nutrient-rich and –poor conditions at 4 °C and infection of human gut epithelial cells was assessed by Tn-insertion site sequencing (Tn-seq). A total of 331 homologous gene clusters were essential for fitness during in vitro growth in three C. jejuni strains, revealing that a large part of its genome is dedicated to growth. We report novel C. jejuni factors essential throughout its life cycle. Importantly, we identified genes that fulfil important roles across multiple conditions. Our comprehensive screens showed which flagella elements are essential for growth and which are vital to the interaction with host organisms. Future efforts should focus on how to exploit this knowledge to effectively control infections caused by C. jejuni.

Genomic variations leading to alterations in cell morphology of Campylobacter spp

Campylobacter jejuni, the most common cause of bacterial diarrhoeal disease, is normally helical. However, it can also adopt straight rod, elongated helical and coccoid forms. Studying how helical morphology is generated, and how it switches between its different forms, is an important objective for understanding this pathogen. Here, we aimed to determine the genetic factors involved in generating the helical shape of Campylobacter. A C. jejuni transposon (Tn) mutant library was screened for non-helical mutants with inconsistent results. Whole genome sequence variation and morphological trends within this Tn library, and in various C. jejuni wild type strains, were compared and correlated to detect genomic elements associated with helical and rod morphologies. All rod-shaped C. jejuni Tn mutants and all rod-shaped laboratory, clinical and environmental C. jejuni and Campylobacter coli contained genetic changes within the pgp1 or pgp2 genes, which encode peptidoglycan modifying enzymes. We therefore confirm the importance of Pgp1 and Pgp2 in the maintenance of helical shape and extended this to a wide range of C. jejuni and C. coli isolates. Genome sequence analysis revealed variation in the sequence and length of homopolymeric tracts found within these genes, providing a potential mechanism of phase variation of cell shape.

Structure–function analysis of the Bacillus megaterium GerUD spore germinant receptor protein

Germination of Bacillus spores is triggered by the interaction of germinant molecules with specialized receptor proteins localized to the spore inner membrane. Germinant receptors (GRs) are comprised typically of three interacting protein subunits, each of which is essential for receptor function. At least some GRs appear to have a fourth component, referred to as a D-subunit protein. A number of D-subunit proteins were shown previously to be capable of modulating the activity of associated GRs. Here, we investigate the topology and structure–function relationships of the Bacillus megaterium QM B1551 GerUD protein, which is associated with the GerU GR. The presented data demonstrate that GerUD can be subjected to relatively extensive structural modifications while retaining function. Indeed, the presence of either of the two transmembrane spanning domains is sufficient to modulate an efficient GerU-mediated germinative response. The precise function of D-subunit proteins has yet to be established, although they may act as molecular chaperones within the spore inner-membrane environment.

Phylogenetic analyses and antimicrobial resistance profiles of Campylobacter spp. from diarrhoeal patients and chickens in Botswana

Campylobacter spp. are a leading cause of bacterial enteritis worldwide, including countries in Africa, and have been identified by the World Health Organisation (WHO) as one of the high priority antimicrobial resistant pathogens. However, at present there is little knowledge on the prevalence, molecular epidemiology or antimicrobial susceptibility of Campylobacter spp. isolates in Botswana, both in patients and in the zoonotic context. Some data indicate that ~14% of diarrhoeal disease cases in a paediatric setting can be ascribed to Campylobacter spp., urging the need for the magnitude of Campylobacter-associated diarrhoea to be established. In this survey, we have characterised the genomic diversity of Campylobacter spp. circulating in Botswana isolated from cases of diarrhoeal disease in humans (n = 20) and from those that colonised commercial broiler (n = 35) and free-range (n = 35) chickens. Phylogeny showed that the Campylobacter spp. isolated from the different poultry and human sources were highly related, suggesting that zoonotic transmission has likely occurred. We found that for Campylobacter spp. isolated from humans, broilers and free-range chickens, 52% was positive for tetO, 47% for gyrA-T86I, 72% for blaOXA-61, with 27% carrying all three resistance determinants. No 23S mutations conferring macrolide resistance were detected in this survey. In summary, our study provides insight into Campylobacter spp. in poultry reservoirs and in diarrhoeal patients, and the relevance for treatment regimens in Botswana.

Identification and initial characterisation of a protein involved in Campylobacter jejuni cell shape.

Campylobacter jejuni is the leading cause of bacterial food borne illness. While helical cell shape is considered important for C. jejuni pathogenesis, this bacterium is capable of adopting other morphologies. To better understand how helical-shaped C. jejuni maintain their shape and thus any associated colonisation, pathogenicity or other advantage, it is first important to identify the genes and proteins involved. So far, two peptidoglycan modifying enzymes Pgp1 and Pgp2 have been shown to be required for C. jejuni helical cell shape. We performed a visual screen of ∼2000 transposon mutants of C. jejuni for cell shape mutants. Whole genome sequence data of the mutants with altered cell shape, directed mutants, wild type stocks and isolated helical and rod-shaped ‘wild type’ C. jejuni, identified a number of different mutations in pgp1 and pgp2, which result in a change in helical to rod bacterial cell shape. We also identified an isolate with a loss of curvature. In this study, we have identified the genomic change in this isolate, and found that targeted deletion of the gene with the change resulted in bacteria with loss of curvature. Helical cell shape was restored by supplying the gene in trans. We examined the effect of loss of the gene on bacterial motility, adhesion and invasion of tissue culture cells and chicken colonisation, as well as the effect on the muropeptide profile of the peptidoglycan sacculus. Our work identifies another factor involved in helical cell shape.

Genome and Plasmid Sequences of Escherichia coli KV7, an Extended-Spectrum β-Lactamase Isolate Derived from Feces of a Healthy Pig

ABSTRACT We present single-contig assemblies for Escherichia coli strain KV7 (serotype O27, phylogenetic group D) and its six plasmids, isolated from a healthy pig, as determined by PacBio RS II and Illumina MiSeq sequencing. The chromosome of 4,997,475 bp and G+C content of 50.75% harbored 4,540 protein-encoding genes.

The GerW Protein Is Not Involved in the Germination of Spores of Bacillus Species

Germination of dormant spores of Bacillus species is initiated when nutrient germinants bind to germinant receptors in spores’ inner membrane and this interaction triggers the release of dipicolinic acid and cations from the spore core and their replacement by water. Bacillus subtilis spores contain three functional germinant receptors encoded by the gerA, gerB, and gerK operons. The GerA germinant receptor alone triggers germination with L-valine or L-alanine, and the GerB and GerK germinant receptors together trigger germination with a mixture of L-asparagine, D-glucose, D-fructose and KCl (AGFK). Recently, it was reported that the B. subtilis gerW gene is expressed only during sporulation in developing spores, and that GerW is essential for L-alanine germination of B. subtilis spores but not for germination with AGFK. However, we now find that loss of the B. subtilis gerW gene had no significant effects on: i) rates of spore germination with L-alanine; ii) spores’ levels of germination proteins including GerA germinant receptor subunits; iii) AGFK germination; iv) spore germination by germinant receptor-independent pathways; and v) outgrowth of germinated spores. Studies in Bacillus megaterium did find that gerW was expressed in the developing spore during sporulation, and in a temperature-dependent manner. However, disruption of gerW again had no effect on the germination of B. megaterium spores, whether germination was triggered via germinant receptor-dependent or germinant receptor-independent pathways.