Pectobacterium parmentieri SCC 3193 Mutants with Altered Synthesis of Cell Surface Polysaccharides Are Resistant to N4-Like Lytic Bacteriophage ϕA38 (vB_Ppp_A38) but Express Decreased Virulence in Potato (Solanum tuberosum L.) Plants.

Pectobacterium parmentieri is a Gram-negative plant-pathogenic bacterium able to infect potato (Solanum tuberosum L.). Little is known about lytic bacteriophages infecting P. parmentieri and how phage-resistance influences the environmental fitness and virulence of this species. A lytic phage vB_Ppp_A38 (ϕA38) has been previously isolated and characterized as a potential biological control agent for the management of P. parmentieri. In this study, seven P. parmentieri SCC 3193 Tn5 mutants were identified that exhibited resistance to infection caused by vB_Ppp_A38 (ϕA38). The genes disrupted in these seven mutants encoded proteins involved in the assembly of O-antigen, sugar metabolism, and the production of bacterial capsule exopolysaccharides. The potential of A38-resistant P. parmentieri mutants for plant colonization and pathogenicity as well as other phenotypes expected to contribute to the ecological fitness of P. parmentieri, including growth rate, use of carbon and nitrogen sources, production of pectinolytic enzymes, proteases, cellulases, and siderophores, swimming and swarming motility, presence of capsule and flagella as well as the ability to form biofilm were assessed. Compared to the wild-type P. parmentieri strain, all phage-resistant mutants exhibited a reduced ability to colonize and to cause symptoms in growing potato (S. tuberosum L.) plants. The implications of bacteriophage resistance on the ecological fitness of P. parmentieri are discussed.

Bifidobacterium breve UCC2003 Exopolysaccharide Modulates the Early Life Microbiota by Acting as a Potential Dietary Substrate.

BACKGROUND: Bifidobacterium represents an important early life microbiota member. Specific bifidobacterial components, exopolysaccharides (EPS), positively modulate host responses, with purified EPS also suggested to impact microbe-microbe interactions by acting as a nutrient substrate. Thus, we determined the longitudinal effects of bifidobacterial EPS on microbial communities and metabolite profiles using an infant model colon system. METHODS: Differential gene expression and growth characteristics were determined for each strain; Bifidobacterium breve UCC2003 and corresponding isogenic EPS-deletion mutant (B. breve UCC2003del). Model colon vessels were inoculated with B. breve and microbiome dynamics monitored using 16S rRNA sequencing and metabolomics (NMR). RESULTS: Transcriptomics of EPS mutant vs. B. breve UCC2003 highlighted discrete differential gene expression (e.g., eps biosynthetic cluster), though overall growth dynamics between strains were unaffected. The EPS-positive vessel had significant shifts in microbiome and metabolite profiles until study end (405 h); with increases of Tyzzerella and Faecalibacterium, and short-chain fatty acids, with further correlations between taxa and metabolites which were not observed within the EPS-negative vessel. CONCLUSIONS: These data indicate that B. breve UCC2003 EPS is potentially metabolized by infant microbiota members, leading to differential microbial metabolism and altered metabolite by-products. Overall, these findings may allow development of EPS-specific strategies to promote infant health.

Invasive meningococcal disease in patients with complement deficiencies: a case series (2008-2017).

BACKGROUND: To describe patients with inherited and acquired complement deficiency who developed invasive meningococcal disease (IMD) in England over the last decade. METHODS: Public Health England conducts enhanced surveillance of IMD in England. We retrospectively identified patients with complement deficiency who developed IMD in England during 2008-2017 and retrieved information on their clinical presentation, vaccination status, medication history, recurrence of infection and outcomes, as well as characteristics of the infecting meningococcal strain. RESULTS: A total of 16 patients with 20 IMD episodes were identified, including four with two episodes. Six patients had inherited complement deficiencies, two had immune-mediated conditions associated with complement deficiency (glomerulonephritis and vasculitis), and eight others were on Eculizumab therapy, five for paroxysmal nocturnal haemoglobinuria and three for atypical haemolytic uraemic syndrome. Cultures were available for 7 of 11 episodes among those with inherited complement deficiencies/immune-mediated conditions and the predominant capsular group was Y (7/11), followed by B (3/11) and non-groupable (1/11) strains. Among patients receiving Eculizumab therapy, 3 of the 9 episodes were due to group B (3/9), three others were NG but genotypically group B, and one case each of groups E, W and Y. CONCLUSIONS: In England, complement deficiency is rare among IMD cases and includes inherited disorders of the late complement pathway, immune-mediated disorders associated with low complement levels and patients on Eculizumab therapy. IMD due to capsular group Y predominates in patient with inherited complement deficiency, whilst those on Eculizumab therapy develop IMD due to more diverse capsular groups including non-encapsulated strains.

Phosphorus limitation increases attachment in Agrobacterium tumefaciens and reveals a conditional functional redundancy in adhesin biosynthesis.

Bacterial responses to phosphorus limitation, commonly inorganic phosphate (P(i)), are important survival mechanisms in a variety of environments. The two-component sensor kinase PhoR and its cognate response regulator PhoB are central to the P(i) limitation response of many bacteria and control the large Pho regulon. Limitation for P(i) significantly increased attachment and biofilm formation by the plant pathogen Agrobacterium tumefaciens, and this was driven by PhoB. Surprisingly, it was also found that both phoR and phoB were essential in A. tumefaciens. Expression of a plasmid-borne copy of the low affinity P(i) transporter (pit) from Sinorhizobium meliloti in A. tumefaciens abolished the phoB and phoR essentiality in A. tumefaciens and allowed direct demonstration of the requirement for this regulatory system in the biofilm response. Increased attachment under P(i) limitation required a unipolar polysaccharide (UPP) adhesin. Mutation of a polyisoprenylphosphate hexose-1-phosphate transferase (PHPT) called uppE abolished UPP production and prevented surface attachment under P(i)-replete conditions, but this was rescued under P(i) limitation, and this rescue required phoB. In low P(i) conditions, either uppE or a paralogous gene Atu0102 is functionally redundant, but only uppE functions in UPP synthesis and attachment when P(i) is replete. This conditional functional redundancy illustrates the influence of phosphorus availability on A. tumefaciens surface colonization.

Plant host and sugar alcohol induced exopolysaccharide biosynthesis in the Burkholderia cepacia complex.

The species that presently constitute the Burkholderia cepacia complex (Bcc) have multiple roles; they include soil and water saprophytes, bioremediators, and plant, animal and human pathogens. Since the first description of pathogenicity in the Bcc was based on sour skin rot of onion bulbs, this study returned to this plant host to investigate the onion-associated phenotype of the Bcc. Many Bcc isolates, which were previously considered to be non-mucoid, produced copious amounts of exopolysaccharide (EPS) when onion tissue was provided as the sole nutrient. EPS production was not species-specific, was observed in isolates from both clinical and environmental sources, and did not correlate with the ability to cause maceration of onion tissue. Chemical analysis suggested that the onion components responsible for EPS induction were primarily the carbohydrates sucrose, fructose and fructans. Additional sugars were investigated, and all alcohol sugars tested were able to induce EPS production, in particular mannitol and glucitol. To investigate the molecular basis for EPS biosynthesis, we focused on the highly conserved bce gene cluster thought to be involved in cepacian biosynthesis. We demonstrated induction of the bce gene cluster by mannitol, and found a clear correlation between the inability of representatives of the Burkholderia cenocepacia ET12 lineage to produce EPS and the presence of an 11 bp deletion within the bceB gene, which encodes a glycosyltransferase. Insertional inactivation of bceB in Burkholderia ambifaria AMMD results in loss of EPS production on sugar alcohol media. These novel and surprising insights into EPS biosynthesis highlight the metabolic potential of the Bcc and show that a potential virulence factor may not be detected by routine laboratory culture. Our results also highlight a potential hazard in the use of inhaled mannitol as an osmolyte to improve mucociliary clearance in individuals with cystic fibrosis.

Further characterization of Vibrio vulnificus rugose variants and identification of a capsular and rugose exopolysaccharide gene cluster.

Capsular polysaccharide (CPS) is a major virulence factor in Vibrio vulnificus, and encapsulated strains have an opaque, smooth (OpS) colony morphology, while nonencapsulated strains have a translucent, smooth (TrS) colony morphology. Previously, we showed that OpS and TrS parental strains can yield a third colony type, rugose (R), and that the resulting strains, with the OpR and TrR phenotypes, respectively, form copious biofilms. Here we show that while OpR and TrR strains both produce three-dimensional biofilm structures that are indicative of rugose extracellular polysaccharide (rEPS) production, OpR strains also retain expression of CPS and are virulent in an iron-supplemented mouse model, while TrR strains lack CPS and are avirulent. Chlorine resistance assays further distinguished OpR and TrR isolates as exposure to 3 microg/ml NaOCl eradicated both OpS and OpR strains, while both TrS and TrR strains survived, but at rates which were significantly different from one another. Taken together, these results further emphasize the importance of CPS for virulence of V. vulnificus and establish a correlation between CPS expression and chlorine sensitivity in this organism. Using reverse transcriptase PCR, we also identified a nine-gene cluster associated with both CPS and rEPS expression in V. vulnificus, designated the wcr (capsular and rugose polysaccharide) locus, with expression occurring primarily in R variants. The latter results set the stage for characterization of functional determinants which individually or collectively contribute to expression of multiple EPS forms in this pathogen.

Isolation and characterization of mucous exopolysaccharide (EPS) produced by Vibrio furnissii strain VB0S3.

Marine bacterial strains were isolated from coastal regions of Goa and screened for the strains that produce the highest amount of mucous exopolysaccharide (EPS). Our screening resulted in the identification of the strain Vibrio furnissii VB0S3 (hereafter called VB0S3), as it produced the highest EPS in batch cultures during the late logarithmic growth phase. The isolate was identified as VB0S3 based on morphological and biochemical properties. Growth and EPS production were studied in mineral salts medium supplemented with NaCl (1.5%) and glucose (0.2%). The exopolymer was recovered from the culture supernatant by using three volumes of cold ethanol precipitation and dialysis procedure. Chemical analyses of EPS revealed that it is primarily composed of neutral sugars, uronic acids, and proteins. Fourier-transform infrared (FT-IR) spectroscopy revealed the presence of carboxyl, hydroxyl, and amide groups, which correspond to a typical heteropolymeric polysaccharide, and the EPS also possessed good emulsification activity. The gas chromatographic analysis of an alditol-acetate derivatized sample of EPS revealed that it was mainly composed of galactose and glucose. Minor components found were mannose, rhamnose, fucose, ribose, arabinose, and xylose. EPS was readily isolated from culture supernatants, which suggests that the EPS was a slime-like exopolysaccharide. This is the first report of exopolysaccharide characterization that describes the isolation and characterization of an EPS expressed by Vibrio furnissii strain VB0S3. The results of the study contribute significantly and go a long way towards an understanding of the correlation between growth and EPS production, chemical composition, and industrial applications of the exopolysaccharide in environmental biotechnology and bioremediation.

Structure and role in symbiosis of the exoB gene of Rhizobium leguminosarum bv trifolii.

The Rhizobium leguminosarum bv trifolii exoB gene has been isolated by heterologous complementation of an exoB mutant of R. meliloti. We have cloned a chromosomal DNA fragment from the R. leguminosarum bv trifolii genome that contains an open reading frame of 981 bp showing 80% identity at the amino acid level to the UDP-glucose 4-epimerase of R. meliloti. This enzyme produces UDP-galactose, the donor of galactosyl residues for the lipid-linked oligosaccharide repeat units of various heteropolysaccharides of rhizobia. An R. leguminosarum bv trifolii exoB disruption mutant differed from the wild type in the structure of both the acidic exopolysaccharide and the lipopolysaccharide. The acidic exopolysaccharide made by our wild-type strain is similar to the Type 2 exopolysaccharide made by other R. leguminosarum bv trifolii wild types. The exopolysaccharide made by the exoB mutant lacked the galactose residue and the substitutions attached to it. The exoB mutant induced the development of abnormal root nodules and was almost completely unable to invade plant cells. Our results stress the importance of exoB in the Rhizobium-plant interaction.

Cloning and characterization of four genes of Rhizobium leguminosarum bv. trifolii involved in exopolysaccharide production and nodulation.

Four different genes of Rhizobium leguminosarum bv. trifolii strain RBL5599 involved in exopolysaccharide (EPS) production were identified by complementation of Tn5-induced EPS-deficient mutants (Exo mutants) with a cosmid bank. On one cosmid pssA was located, which was found to be almost identical to the pss4 gene from R. leguminosarum bv. viciae VF39 and highly homologous to a family of glycosyl transferases. Two pssA mutants, exo2 and exo4, were characterized and found to produce 19 and 1% of the wild-type amount of EPS, respectively. The three other genes were found to be closely linked on a different complementing cosmid. pssC revealed similarity to exoM and exoW of R. meliloti, both encoding glucosyl transferases involved in the synthesis of succinoglycan. A mutation in this gene (mutant exo50) did reduce EPS synthesis to 27% of the wild-type amount. We found an operon closely linked to pssC, consisting of two overlapping genes, pssD and pssE, that is essential for EPS production. Homology of pssD and pssE was found with cps14F and cps14G of Streptococcus pneumoniae, respectively: two genes responsible for the second step in capsule polysaccharide synthesis. Furthermore, pssD and pssE were homologous to the 5' and 3' parts, respectively, of spsK of Sphingomonas S88, which encodes a putative glycosyl transferase. Structural analysis of EPS produced by Exo mutants exo2, exo4, and exo50 showed it to be identical to that of the parental strain RBL5599, with the exception of acetyl groups esterified to one of the glucose residues being absent.

Emergence of Cu(++)-tolerant mutants defective in gellan synthesis in Cu(++)-stressed cultures of Sphingomonas paucimobilis.

Cells defective in gellan synthesis appeared during cultivation of the gellan gum-producing strain Sphingomonas paucimobilis R40 with inhibitory concentrations of copper, supplied as CuCl2. The percentage of less mucoid colonial variants dramatically increased with the increase in Cu++ supplementation, reaching 85% of total viable cells at the maximal concentration for growth. Results reported in this work indicate that emergence of colonial variants defective in gellan synthesis results from Cu(++)-induced mutation and the growth advantage of these mutants in Cu(++)-stressed cultures. In fact, DNA homologous recombination strongly increased with the increase in copper supplementation as indicated by the regeneration of kanamycin-resistant cells of R40 harbouring plasmid pBX404-7, which carries two non-overlapping truncated genes derived from a gene conferring kanamycin resistance. The four major groups of colonial mutants that emerged from Cu(++)-stressed cultures of R40 exhibited reduced growth rate and biomass yield in the absence of Cu++ stress and produced decreased levels of exopolysac-charide (EPS) which yielded solutions of lower or negligible viscosity. The level of increased Cu++ tolerance of these mutants, assessed by the inhibitory effect of Cu++ on growth, correlated with the degree of loss of the ability to secrete high-molecular-mass EPS. Consistent with the growth advantage of gellan-defective mutants in Cu(++)-stressed cultures, the non-producing strain RP10, spontaneously obtained during extended cultivation of R40, also exhibited a higher tolerance to Cu++. In addition, its non-mucoid phenotype was stably maintained during Cu(++)-stressed cultivation despite the stimulation of homologous recombination by Cu++.

Influence of pili, fibrils, and capsule on in vitro adherence by Haemophilus influenzae type b.

Haemophilus influenzae type b is an encapsulated bacterium that initiates infection by colonizing the upper respiratory epithelium. In vitro studies indicate that H. influenzae type b is capable of expressing two morphologically distinct filamentous adhesive structures, referred to as pili and fibrils, respectively. In this study, we examined adherence to a variety of human epithelial-cell types and demonstrated that pili and fibrils have separate cellular binding specificities. In addition, we found that capsular material inhibits fibril recognition of the host-cell surface. This inhibitory effect was reduced when bacteria were grown to stationary phase, reflecting diminished encapsulation. However, when growth medium was supplemented with Mg2+, stationary-phase organisms were relatively heavily encapsulated and non-adherent. These observations suggest that encapsulation can be modulated in response to growth phase or environmental signals. It is possible that encapsulation is down-modulated early in the infectious process in order to avoid interfering with colonization. In contrast, encapsulation may be up-modulated between hosts and during bacteremia, where it appears to confer a selective advantage. We speculate that this model may also apply to other encapsulated pathogens.

Cloning of a locus involved in Streptococcus mutans intracellular polysaccharide accumulation and virulence testing of an intracellular polysaccharide-deficient mutant.

The streptococcal transposon Tn916 (Tcr) was used to isolate mutants of Streptococcus mutans altered in glycogen accumulation to investigate whether glycogenlike intracellular polysaccharides (IPS) play an important role in S. mutans-induced caries formation. S. mutans UA130 (serotype c) was transformed with the Escherichia coli plasmid pAM620 (Tn916), and the resultant transposon libraries were screened for glycogen content by iodine staining. A transposon mutant, designated SMS201, demonstrated a glycogen-deficient phenotype on glucose-enriched medium. Quantitative electron microscopy confirmed that IPS concentrations were significantly reduced in SMS201 relative to the wild-type progenitor strain, UA130. Importantly, reversion to wild type correlated at all times with loss of the transposon. Transposon excisants were used to facilitate cloning of the streptococcal gene(s) involved in glycogen biosynthesis and storage. A 2.1-kb chromosomal determinant (glgR) which encodes a putative regulator of S. mutans glycogen accumulation was isolated. A stable deletion mutation (delta glgR) was subsequently generated in E. coli and introduced into S. mutans by allelic exchange. The resultant glycogen synthesis-deficient mutant, SMS203, demonstrated a significantly reduced cariogenic potential (P less than 0.01) on the buccal, sulcal, and proximal surfaces of teeth in germfree rats, relative to animals challenged with the glycogen synthesis-proficient progenitor strain, UA130. These observations confirm previous reports (J. M. Tanzer, M. L. Freedman, F. N. Woodiel, R. L. Eifert, and L. A. Rinehimer, p. 597-616, in H. M. Stiles, W. J. Loesche, and T. L. O'Brien, ed., Proceedings in Microbiology. Aspects of Dental Caries. Special Supplement to Microbiology Abstracts, vol. 3, 1976) which implicate IPS as significant contributors to the S. mutans cariogenic process.

Unusual structure of the exopolysaccharide of Rhizobium leguminosarum bv. viciae strain 248.

The exopolysaccharide from R. leguminosarum bv. viciae strain 248 differs from those of other Rhizobium strains with similar symbiotic behavior. 13C-N.m.r. spectroscopy of fragments generated by partial hydrolysis, together with methylation analysis and 13C-n.m.r. spectroscopy of the enzymically depolymerised exopolysaccharide, indicated the following nonasaccharide repeating-unit: [formula: see text] The locations of the acetyl and 3-hydroxybutanoyl substituents in the exopolysaccharide are assigned provisionally. R. leguminosarum bv. viciae strain 248, cured of its Sym plasmid pRL1JI, synthesised an exopolysaccharide in which the sites and degree of substitution were unchanged. A Tn5 mutant, derived from strain 248 and unable to induce nodules, synthesised small amounts of EPS that lacked galactose.

The region for exopolysaccharide synthesis in Rhizobium leguminosarum biovar trifolii is located on the non-symbiotic plasmid.

An Exo- mutant of Rhizobium leguminosarum biovar trifolii was isolated which did not produce acidic exopolysaccharide and induced defective, non-fixing nodules on clover plants. The nodules were defective at a late stage of development, they contained infection threads and bacteria were released into the host cells. Cosmid pARF136 capable of complementing the Exo- mutation was isolated from a cosmid bank made from total R. trifolii DNA. Hybridization between DNA of pARF136 and plasmids of R. trifolii strains separated by Eckhardt's technique suggested that the exo locus is located on a 300 kb megaplasmid, and nodDABC and nifKDH genes are located on another 180 kb pSym plasmid. A 5.4 kb BamH1 fragment of the recombinant cosmid pARF136 was able to restore exopolysaccharide synthesis in Exo- mutant of R. trifolii 93 but it did not complement the symbiotic defect.

Rhizobium leguminosarum exopolysaccharide mutants: biochemical and genetic analyses and symbiotic behavior on three hosts.

Ten independently generated mutants of Rhizobium leguminosarum biovar phaseoli CFN42 isolated after Tn5 mutagenesis formed nonmucoid colonies on all agar media tested and lacked detectable production of the normal acidic exopolysaccharide in liquid culture. The mutants were classified into three groups. Three mutants harbored Tn5 insertions on a 3.6-kilobase-pair EcoRI fragment and were complemented to have normal exopolysaccharide production by cosmids that shared an EcoRI fragment of this size from the CFN42 genome. The Tn5 inserts of five other mutants appeared to be located on a second, slightly smaller EcoRI fragment. Attempts to complement mutants of this second group with cloned DNA were unsuccessful. The mutations of the other two mutants were located in apparently adjacent EcoRI fragments carried on two cosmids that complemented those two mutants. The latter two mutants also lacked O-antigen-containing lipopolysaccharides and induced underdeveloped nodules that lacked nitrogenase activity on bean plants. The other eight mutants had normal lipopolysaccharides and wild-type symbiotic proficiencies on bean plants. Mutants in each of these groups were mated with R. leguminosarum strains that nodulated peas (R. leguminosarum biovar viciae) or clovers (R. leguminosarum biovar trifolii). Transfer of the Tn5 mutations resulted in exopolysaccharide-deficient R. leguminosarum biovar viciae or R. leguminosarum biovar trifolii transconjugants that were symbiotically deficient in all cases. These results support earlier suggestions that successful symbiosis with peas or clovers requires that rhizobia be capable of acidic exopolysaccharide production, whereas symbiosis with beans does not have this requirement.

Symbiotic nitrogen fixation: molecular cloning of Rhizobium genes involved in exopolysaccharide synthesis and effective nodulation.

A transposon (Tn5)-induced mutant (strain ANU437) of Rhizobium trifolii was isolated in which no water-soluble exopolysaccharide (EPS) could be detected. This mutant was also incapable of forming nitrogen-fixing root nodules on clover plants. Molecular cloning has demonstrated that the Tn5 transposon was responsible for both of these mutant phenotypes and that there is a direct correlation between EPS synthesis in this bacterial strain and its ability to carry out symbiotic nitrogen fixation. In the mutant ANU437, Tn5 was located in a 9.4-kb EcoRI fragment that was cloned into the amplifiable plasmid pBR322. The recombinant plasmid was used as a hybridization probe to isolate the corresponding wild-type DNA sequence of R. trifolii from a lambda Charon 28 genomic clone bank. This DNA sequence was subcloned into the broad host range conjugative plasmid RP4 and introduced into the Escherichia coli strain RR1. It was then transferred to the mutant ANU437 by conjugation. The acquisition of the wild-type DNA sequence by the mutant ANU437 resulted in the restoration of its ability to synthesize normal levels of EPS and to form nitrogen-fixing nodules on white and subterranean clovers.