Outer Membrane Protein Profiles Research Articles

Comparison of the Transport of Tetracycline-Resistant and Tetracycline-Susceptible Escherichia coli Isolated from Lake Michigan

It was recently reported that tetracycline could enhance the mobility of manure-derived Escherichia coli within saturated porous media (Walczak et al. (Water Research 45:1681-1690, 2011)). It was also shown, however, that E. coli from various sources could display marked variation in their mobility (Bolster et al. (Journal of Environmental Quality 35:1018-1025, 2009)). The focus of this research was to examine if the observed difference in the mobility of manure-derived tetracycline-resistant (tet(R)) and tetracycline-susceptible (tet(S)) E. coli strains was source-dependent. Specifically, E. coli were isolated from Lake Michigan, and the influence of tetracycline resistance on Lake Michigan-derived E. coli was investigated through column transport experiments. Additionally, a variety of cell morphology and surface properties were determined and related to the observed bacterial transport behavior. Our experimental results showed that, consistent with previous observations, the deposition rate coefficients of the tet(R)E. coli strain was ~20-100% higher than those of the tet(S)E. coli strain. The zeta potential of the tet(R)E. coli cells was ~25 mV more negative than the tet(S)E. coli cells. Because the surfaces of the E. coli cells and the quartz sands were negatively charged, the repulsive electrostatic double-layer interaction between the tet(R)E. coli cells and the quartz sands was stronger, and the mobility of the tet(R)E. coli cells in the sand packs was thus higher. The tet(R)E. coli cells were also more hydrophilic than the tet(S)E. coli cells. Results from migration to hydrocarbon phase (MATH) tests showed that about ~35% more tet(S)E. coli cells partitioned to the hydrocarbon phase. As it was previously shown that cell hydrophobicity could enhance the attachment of bacterial cells to quartz sand, the difference in cell hydrophobicity could also have contributed to the observed higher mobility of the tet(R)E. coli cells. The size of the tet(R) and tet(S)E. coli cells were similar, suggesting that the observed difference in their mobility was not size-related. Characterization of cell surface properties also showed that tet(R) and tetS E. coli cells differed slightly in cell-bound lipopolysaccharide contents and had distinct outer membrane protein profiles. Such difference could alter cell surface properties which in turn led to changes in cell mobility.

Phenotypical and genetic characterization of Yersinia ruckeri strains isolated from recent outbreaks in farmed rainbow trout Oncorhynchus mykiss (Walbaum) in Peru

A total of 30 strains of Yersinia ruckeri causing recent outbreaks in Peruvian trout culture systems, were studied by means of biochemical characteristics, serology, lipopolysaccharide (LPS) and outer membrane protein (OMP) analysis, and ERIC and REP PCR fingerprinting. All the Peruvian isolates were found to be fermentative, oxidase negative and positive for decarboxylation of lysine and ornithine and utilization of glucose and mannitol, allowing their presumptive identification as Y. ruckeri. Sequencing of the 16S rRNA gene confirmed that isolates were indeed Y. ruckeri (> 99.98% identity). Although most of the strains studied were motile and lipase positive corresponding to the biotype 1 of Y. ruckeri, 5 of these strains were negative from both tests, being identified as biotype 2. In addition, drug susceptibility tests determined high sensitivity to sulfamethoxazole/trimethoprim, oxytetracycline, ampicillin and enrofloxacin in all the isolates. Serologically, all the Peruvian strains studied were identified as belonging to the serotype O1 subgroup a. Analysis of the lipopolysaccharide (LPS) as well as total and outer membrane proteins (OMPs) profiles and the correspondent inmunoblotting, supported these results. Genotyping performed by means of ERIC- and REP-PCR determined major correlation of the Peruvian isolates with the type strain NCIMB 2194 T regardless of the biotype.

Deletion of mtrC in Haemophilus ducreyi Increases Sensitivity to Human Antimicrobial Peptides and Activates the CpxRA Regulon

Haemophilus ducreyi resists killing by antimicrobial peptides encountered during human infection, including cathelicidin LL-37, α-defensins, and β-defensins. In this study, we examined the role of the proton motive force-dependent multiple transferable resistance (MTR) transporter in antimicrobial peptide resistance in H. ducreyi. We found a proton motive force-dependent effect on H. ducreyi's resistance to LL-37 and β-defensin HBD-3, but not α-defensin HNP-2. Deletion of the membrane fusion protein MtrC rendered H. ducreyi more sensitive to LL-37 and human β-defensins but had relatively little effect on α-defensin resistance. The mtrC mutant 35000HPmtrC exhibited phenotypic changes in outer membrane protein profiles, colony morphology, and serum sensitivity, which were restored to wild type by trans-complementation with mtrC. Similar phenotypes were reported in a cpxA mutant; activation of the two-component CpxRA regulator was confirmed by showing transcriptional effects on CpxRA-regulated genes in 35000HPmtrC. A cpxR mutant had wild-type levels of antimicrobial peptide resistance; a cpxA mutation had little effect on defensin resistance but led to increased sensitivity to LL-37. 35000HPmtrC was more sensitive than the cpxA mutant to LL-37, indicating that MTR contributed to LL-37 resistance independent of the CpxRA regulon. The CpxRA regulon did not affect proton motive force-dependent antimicrobial peptide resistance; however, 35000HPmtrC had lost proton motive force-dependent peptide resistance, suggesting that the MTR transporter promotes proton motive force-dependent resistance to LL-37 and human β-defensins. This is the first report of a β-defensin resistance mechanism in H. ducreyi and shows that LL-37 resistance in H. ducreyi is multifactorial.

Comparative proteogenomic analysis of the Leptospira interrogans virulence-attenuated strain IPAV against the pathogenic strain 56601

The virulence-attenuated Leptospira interrogans serovar Lai strain IPAV was derived by prolonged laboratory passage from a highly virulent ancestral strain isolated in China. We studied the genetic variations of IPAV that render it avirulent via comparative analysis against the pathogenic L. interrogans serovar Lai strain 56601. The complete genome sequence of the IPAV strain was determined and used to compare with, and then rectify and reannotate the genome sequence of strain 56601. Aside from their highly similar genomic structure and gene order, a total of 33 insertions, 53 deletions and 301 single-nucleotide variations (SNVs) were detected throughout the genome of IPAV directly affecting 101 genes, either in their 5' upstream region or within their coding region. Among them, the majority of the 44 functional genes are involved in signal transduction, stress response, transmembrane transport and nitrogen metabolism. Comparative proteomic analysis based on quantitative liquid chromatography (LC)-MS/MS data revealed that among 1 627 selected pairs of orthologs, 174 genes in the IPAV strain were upregulated, with enrichment mainly in classes of energy production and lipid metabolism. In contrast, 228 genes in strain 56601 were upregulated, with the majority enriched in the categories of protein translation and DNA replication/repair. The combination of genomic and proteomic approaches illustrated that altered expression or mutations in critical genes, such as those encoding a Ser/Thr kinase, carbon-starvation protein CstA, glutamine synthetase, GTP-binding protein BipA, ribonucleotide-diphosphate reductase and phosphate transporter, and alterations in the translational profile of lipoproteins or outer membrane proteins are likely to account for the virulence attenuation in strain IPAV.

Influence of tetracycline resistance on the transport of manure-derived Escherichia coli in saturated porous media

In this research, tetracycline resistant (tet R) and tetracycline susceptible (tet S) Escherichia coli isolates were retrieved from dairy manure and the influence of tetracycline resistance on the transport of E. coli in saturated porous media was investigated through laboratory column transport experiments. Experimental results showed that tet R E. coli strains had higher mobility than the tet S strains in saturated porous media. Measurements of cell surface properties suggested that tet R E. coli strains exhibited lower zeta potentials than the tet S strains. Because the surface of clean quartz sands is negatively charged, the repulsive electrostatic double layer (EDL) interaction between the tet R cells and the surface of sands was stronger and thus facilitated the transport of the tet R cells. Although no difference was observed in surface acidity, cell size, lipopolysaccharides (LPS) sugar content and cell-bound protein levels between the tet R and tet S strains, they displayed distinct outer membrane protein (OMP) profiles. It was likely that the difference in OMPs, some potentially related to drug efflux pumps, between the tet R and tet S strains led to alteration in cell surface properties which in turn affected cell transport in saturated porous media. Findings from this research suggested that manure-derived tet R E. coli could spread more widely in the groundwater system and pose serious public health risks.

Identification of outer membrane proteins of Vibrio parahaemolyticus and Vibrio alginolyticus altered in response to γ-irradiation or long-term starvation

Vibrio parahaemolyticus and Vibrio alginolyticus were subjected to γ-irradiation (0.5 kGy) or starvation by incubation for 8 months in seawater to study modifications in their outer membrane protein patterns. After treatment, outer membrane protein profiles of starved or γ-irradiated bacteria were found to be altered when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Altered proteins were identified by mass spectrometry (MS and MS/MS) and analyses revealed that OmpU can be considered a starvation stress-induced protein. In addition, expression of OtnA, OmpW, OmpA and peptidoglycan-associated lipoprotein decreased to non-detectable levels in starved cells. Furthermore, MltA-interacting protein MipA appeared under γ-irradiation or starvation conditions. Thus, it can be considered to be a γ-irradiation, long-term starvation stress protein in some vibrios.

Comparison of Genotypic and Phenotypic Characterization Methods for Pasteurella Multocida Isolates from Fatal Cases of Bovine Respiratory Disease

Bovine respiratory disease (BRD) is the most costly disease of beef cattle in North America. Because Pasteurella multocida is a commensal of the upper respiratory tract, it is generally considered an opportunistic pathogen. However, studies in swine indicated that there may be a limited number of strains associated with disease, suggesting that some are more virulent than others. Although this may also be true of isolates from cattle, appropriate typing methods must be established before this possibility can be investigated. The purpose of this study was to compare effectiveness of polymerase chain reaction (PCR) fingerprinting to more traditional approaches for typing bovine P. multocida isolates. Isolates were obtained from 41 cases of fatal BRD and subjected to random amplified polymorphic DNA PCR (RAPD-PCR), whole cell protein (WCP) profiles, outer membrane protein (OMP) profiles, and serotyping. The discrimination index was calculated for each typing method and combinations of each using Simpson's index of diversity. Correlation coefficients were calculated to assess concordance between classification results achieved through genotypic (RAPD-PCR) and phenotypic (WCP, OMP, and serotyping) approaches. All characterization methods were capable of discriminating between isolates. However, there was poor concordance between techniques. There were also few significant associations between typing results and epidemiologic data. Random amplified polymorphic DNA PCR was validated as being a repeatable and reliable means of discriminating between P. multocida isolates obtained from cattle. Isolates obtained from fatal cases of BRD in calves in a commercial feedlot demonstrated significant diversity, justifying additional investigation into whether P. multocida is a strictly opportunistic pathogen in cattle.

Analysis of Salmonella enteritidis Outer Membrane Proteins and Lipopolysaccharide Profiles with the Detection of Immune Dominant Proteins

Problem statement: Salmonella infection is a serious medical and veterinary problem world-wide and causes great concern in the human health and food safety. Salmonella enteritidis outer membrane proteins and lipopolysaccharide play important role in the virulence and immunological properties of bacteria. Prophylaxis of enterobacterial infection has proven difficult to achieve. Vaccination is an effective tool for the prevention of Salmonella infection. Approach: Therefore, a study was conducted on S. enteritidis isolates from chicken meat samples for Outer Membrane Proteins (OMPs) and Lipopolysaccharide (LPS) profile were analyzed by SDS-PAGE electrophoresis and immunodominent antigen was detected by western blot analysis. Results: S. enteritidis with different OMPs bands were exhibited with molecular weight ranging from 5-90 kDa. The major outer membrane protein profiles of all S. enteritidis isolates were homogenous with different expression in intensity of protein was observed. LPS of different S. enteritidis isolates exhibited doublet band was observed and identified as R type of strains. The immunoblotting results at 14.4 and 24 KDa proteins were good immunogen. Conclusion: The 14.4 and 24 KDa proteins were immune response protein. This proteins can use for vaccine development.

Cellular components of probiotics control Yersinia ruckeri infection in rainbow trout, Oncorhynchus mykiss (Walbaum)

Subcellular components of the probiotics Aeromonas sobria GC2 and Bacillus subtilis JB-1, when administered to rainbow trout, Oncorhynchus mykiss, conferred protection against a new biogroup of Yersinia ruckeri. Thus, intraperitoneal or intramuscular injection of rainbow trout with cell wall proteins (CWPs), outer membrane proteins (OMPs), lipopolysaccharides (LPS), whole cell proteins (WCPs) and live cells followed by challenge on day 8 with Y. ruckeri led to 80-100% survival compared with 10% survival in the controls. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of WCPs and OMPs from GC2 had 10 and 5 variable protein bands in comparison to 11 and 5 bands in the WCPs and CWPs from JB-1. Proteomic analyses were employed following SDS-PAGE to categorize one dominant protein of 104.7 kDa from the CWPs of JB-1 and equated it with 'Bacillus spp. endoglucanase' with a Mascot score >69. These results point to the potential of using cellular components of probiotics for protection of fish against bacterial diseases.

Emergence of Carbapenem-Resistant Escherichia Coli in Taiwan: Resistance Due to Combined CMY-2 Production and Porin Deficiency

Eight pairs of Escherichia Coli isolates with various carbapenem susceptibilities from 8 patients were prospectively collected to study the development of resistance. All carbapenem-resistant E. coli isolates were resistant to all tested β-lactams antibiotics except tigecycline. identical pulsed-field gel electrophoresis (PFGE) patterns were found in carbapenem-susceptible and -resistant isolates but different PFGE patterns occurred among patients. A CMY-2 β-lactamase was found in all E. coli isolates. No previously reported carbapenemase genes were detected. Examination of outer membrane protein (OMP) profiles revealed that OmpA was not found in all isolates, while OmpC and Ompf were lost in carbapenem-resistant isolates. loss of both OmpC and Ompf represents the major mechanism of the development of carbapenem resistance in those patients with CMY-2-producing E. coli infections.

Mass spectrometric characterization of theCampylobacter jejuniadherence factor CadF reveals post-translational processing that removes immunogenicity while retaining fibronectin binding

Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins, such as fibronectin (Fn). Fn-binding is mediated by a 37 kDa outer membrane protein termed Campylobacter adherence Factor (CadF). The outer membrane protein profile of a recent gastrointestinal C. jejuni clinical isolate (JHH1) was analysed using 2-DE and MS. Several spots were identified as products of the cadF gene. These included mass and pI variants of 34 and 30 kDa, as well as 24 kDa (CadF(24)) and 22 kDa (CadF(22)) mass variants. CadF variants were fully characterized by MALDI-TOF MS and MALDI-MS/MS. These data confirmed that CadF forms re-folding variants resulting in spots with lower mass and varying pI that are identical at the amino acid sequence level and are not modified post-translationally. CadF(22) and CadF(24), however, were characterized as N-terminal, membrane-associated polypeptides resulting from cleavage between serine(195) and leucine(196), and glycine(201) and phenylalanine(202), respectively. These variants were more abundant in the virulent (O) isolate of C. jejuni NCTC11168 when compared with the avirulent (genome sequenced) isolate. Hexahistidine fusion constructs of full-length CadF (34 kDa), CadF(24), and the deleted C-terminal OmpA domain (14 kDa; CadF(14)) were created in Escherichia coli. Recombinant CadF variants were probed against patient sera and revealed that only full-length CadF retained reactivity. Binding assays showed that CadF(24) retained Fn-binding capability, while CadF(14) did not bind Fn. These data suggest that the immunogenic epitope of CadF is cleaved to generate smaller Fn-binding polypeptides, which are not recognized by the host humoral response. CadF cleavage therefore may be associated with virulence in C. jejuni.

Effect of Thyme (Thymus vulgaris L.) Essential Oil and Its Main Constituents on the Outer Membrane Protein Composition of Erwinia Strains Studied with Microfluid Chip Technology

Changes in the composition of bacterial outer membrane proteins may result in antibiotic resistance and in altered invasive ability. A rapid method (microfluid chip technology) was used in this study to analyze the outer membrane protein profile changes of Erwinia strains under antibiotic effect and to compare it with the effect of essential oil and its constituents. Streptomycin, gentamicin and thyme essential oil were capable of significantly changing the protein profile. Neither of the main constituents of the essential oil (thymol and carvacrol) can be exclusively responsible for the changes induced. Quantitative changes in the protein profile may contribute to the explanation of antibacterial effect of thyme oil on pathogenic Erwinia. strains.

Mutation of PEB4 alters the outer membrane protein profile ofCampylobacter jejuni

Campylobacter jejuni is a significant cause of human gastroenteritis worldwide. In an attempt to further define bacterial factors that influence infectivity, Cj0596 was identified as playing a role in C. jejuni virulence. Cj0596 is a periplasmic chaperone that is similar to proteins involved in outer membrane protein (OMP) folding in other bacteria. Mutation of cj0596 caused an alteration in the levels of eight OMPs, compared with wild-type bacteria. Replacement of the cj0596 mutation with the wild-type cj0596 gene restored a wild-type OMP profile. The altered OMP profile in the cj0596 mutant was accompanied by significant changes in several virulence properties, including an increase in the ability to autoagglutinate, increased susceptibility to several antimicrobial agents, and increased biofilm formation. In summary, mutation of cj0596 alters the C. jejuni OMP profile and leads to changes in OMP-related phenotypes involved in C. jejuni pathogenesis.

Identification and pathogenicity to rainbow trout, Oncorhynchus mykiss (Walbaum), of some aeromonads

Twelve strains of fish pathogenic aeromonads were identified by 16S rRNA sequencing as Aeromonas bestiarum, A. hydrophila, A. hydrophila subsp. dhakensis, A. salmonicida subsp. salmonicida, A. sobria biovar sobria and A. veronii biovar sobria. Following intramuscular injection, A. hydrophila subsp. dhakensis caused dark liquefying, raised furuncle-like lesions in rainbow trout within 48 h. Extracellular products of all cultures contained gelatinase and lecithinase, and most revealed lipase. Congo red absorption and siderophore production was recorded, but not so the suicide phenomenon or slime production. Sodium dodecyl sulphate polyacrylamide gel electrophoresis profile of the outer membrane proteins (OMP) revealed 10-25 bands, of which major bands were seen in the region of 32.5-47.5 and 62-83 kDa. Marked heterogenicity of the OMP and whole cell protein (WCP) profiles within and among the species was observed. Polypeptides of 83-173 kDa were detected in the WCP profile of the cultures, but they were not expressed in OMP fractions.

A Comparative Study of Clinical and Environmental Isolates of Pseudomonas aeruginosa in Terms of Quorum Sensing, Outer Membrane Proteins and Their Ability to Cause Urinary Tract Infection

Pseudomonas aeruginosa, one of the most common causes of nosocomial urinary tract infections, is widely distributed in environments. Environmental and clinical isolates have been compared on the basis of production of virulence factors and genomic diversity. However, no studies are available regarding the comparison of their physiological and morphological characteristics. 15 clinical and environmental isolates of Pseudomonas aeruginosa were screened for quorum sensing molecules and all were found to be producers of varied levels of acyl homoserine lactones (AHL’s). Both the isolates showed comparable biofilm forming ability but revealed different outer membrane protein profile. Clinical isolate was found to be more virulent than environmental isolate on the basis of urine and renal colonization in experimental urinary tract infection mouse model. Histopathology and other pathology index factors like myeloperoxidase (MPO), malondialdehyde (MDA) and reactive nitrogen intermediate (RNI) level were also found to be more in case of animals infected with clinical isolates. In conclusion, despite similar abilities of biofilm formation and production of quorum sensing signal molecules, clinical isolate was found to have selective advantage in establishing and causing pathology in the urinary tract of mice. Our results indicate that pathogenicity potential of Pseudomonas aeruginosa vary depending on the source of strain as well as its outer membrane protein profile.

Outer Membrane Protein STM3031 (Ail/OmpX-Like Protein) Plays a Key Role in the Ceftriaxone Resistance of Salmonella enterica Serovar Typhimurium

Previously, the putative outer membrane protein STM3031 has been correlated with ceftriaxone resistance in Salmonella enterica serovar Typhimurium. In this study, this protein was almost undetectable in the ceftriaxone-susceptible strain 01-4, but its levels were increased in 01-4 isogenic strains for which MICs were higher. The stm3031 gene deletion mutant, R200(Deltastm3031), was generated and showed >64-fold lower ceftriaxone resistance than R200, supporting a key role for STM3031 in ceftriaxone resistance. To investigate which outer membrane protein(s) was associated with resistance, the outer membrane protein profiles of 01-4, R200, and R200(Deltastm3031) were compared proteomically. Nine proteins were identified as altered. The expression levels of AcrA, TolC, STM3031, STM1530, VacJ, and Psd in R200 were increased; those of OmpC, OmpD, and OmpW were decreased. The expression levels of OmpD, OmpW, STM1530, VacJ, and Psd, but not those of OmpC, AcrA, and TolC, in R200(Deltastm3031) were returned to the levels in strain 01-4. Furthermore, the genes' mRNA levels correlated with their protein levels when the three strains were compared. The detection of higher AcrB levels, linked to higher acrB, acrD, and acrF mRNA levels, in strain R200 than in strains 01-4 and R200(Deltastm3031) suggests that AcrB, AcrD, and AcrF participate in ceftriaxone resistance. Taken together with the location of STM3031 in the outer membrane, these results suggest that STM3031 plays a key role in ceftriaxone resistance, probably by reducing permeability via a decreased porin OmpD level and enhancing export via increased AcrD efflux pump activity.

Localization and characterization of VVA0331, a 489-kDa RTX-like protein, in Vibrio vulnificus YJ016

Vibrio vulnificus YJ016 contains three genes encoding proteins homologous to repeats-in-toxin proteins. One of these genes, vva0331, possesses a long open reading frame of 13,971 bp in length and resides on the small chromosome between two gene clusters encoding a type I secretion system and several regulatory proteins, respectively. Bioinformatic analysis revealed that VVA0331 consist of nineteen 87-amino acid repeats, two Arg-Gly-Asp motifs, four cysteine residues, an outer membrane protein domain, a polysaccharide-binding site and several motifs related to cell adhesions. These features are distinct from those of typical repeat-in-toxins and autotransporter adhesins. Real-time quantitative PCR analysis indicates that vva0331 gene expression is activated at 30 degrees C and regulated by iron. In addition, VVA0331 is present primarily in a secreted form as determined by cell fractionation assay and Western blot analysis. No significant difference in Hep2 cell adherence, cytotoxicity, and virulence was observed between the wild type and vva0331 mutant strains. In contrast, these strains exhibited apparently different outer membrane protein profiles, and antiserum raised against C-terminal region of VVA0331 reacted with an 85-kDa outer membrane protein of V. vulnificus YJ016.

Yersinia ruckeri biotype 2 isolates from mainland Europe and the UK likely represent different clonal groups

There have been increased reports of outbreaks of enteric redmouth disease (ERM) caused by Yersinia ruckeri in previously vaccinated salmonids in Europe, with some of these outbreaks being attributed to emergent non-motile, Tween 80-negative, biotype 2 isolates. To gain information about their likely origins and relationships, a geographically and temporally diverse collection of isolates were characterised by serotyping, biotyping, pulsed-field gel electrophoresis (PFGE) and outer membrane protein (OMP) profiling. A total of 44 pulsotypes were identified from 160 isolates by PFGE, using the restriction enzyme NotI. Serotype O1 isolates responsible for ERM in rainbow trout in both the US and Europe, and including biotype 2 isolates, represented a distinct subgroup of similar pulsotypes. Biotype 2 isolates, responsible for outbreaks of the disease in rainbow trout in the UK, Denmark and Spain, had different pulsotypes, suggesting that they represented different clones that may have emerged separately. Danish biotype 2 isolates recovered since 1995 were indistinguishable by PFGE from the dominant biotype 1 clone responsible for the majority of outbreaks in Denmark and the rest of mainland Europe. In contrast, US biotype 2 isolate YRNC10 had an identical pulsotype and OMP profile to UK biotype 2 isolates, suggesting that there had been exchange of these isolates between the UK and the US in the past. UK Atlantic salmon isolates were genetically and serologically diverse, with 12 distinct pulsotypes identified among 32 isolates.

MucR, a novel membrane-associated regulator of alginate biosynthesis in Pseudomonas aeruginosa.

Alginate biosynthesis by Pseudomonas aeruginosa was shown to be regulated by the intracellular second messenger bis-(3'-5')-cyclic-dimeric-GMP (c-di-GMP), and binding of c-di-GMP to the membrane protein Alg44 was required for alginate production. In this study, PA1727, a c-di-GMP-synthesizing enzyme was functionally analyzed and identified to be involved in regulation of alginate production. Deletion of the PA1727 gene in the mucoid alginate-overproducing P. aeruginosa strain PDO300 resulted in a nonmucoid phenotype and an about 38-fold decrease in alginate production; thus, this gene is designated mucR. The mucoid alginate-overproducing phenotype was restored by introducing the mucR gene into the isogenic DeltamucR mutant. Moreover, transfer of the MucR-encoding plasmid into strain PDO300 led to an about sevenfold increase in alginate production, wrinkly colony morphology, increased pellicle formation, auto-aggregation, and the formation of highly structured biofilms as well as the inhibition of swarming motility. Outer membrane protein profile analysis showed that overproduction of MucR mediates a strong reduction in the copy number of FliC (flagellin), required for flagellum-mediated motility. Translational reporter enzyme fusions with LacZ and PhoA suggested that MucR is located in the cytoplasmic membrane with a cytosolic C terminus. Deletion of the proposed C-terminal GGDEF domain abolished MucR function. MucR was purified and identified using tryptic peptide fingerprinting and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Overall, experimental evidence was provided suggesting that MucR specifically regulates alginate biosynthesis by activation of alginate production through generation of a localized c-di-GMP pool in the vicinity of Alg44.

Molecular Characterization of Pasteurella multocida Isolates From Fatal Cases of Bovine Respiratory Disease

Bovine respiratory disease (BRD) is the most costly disease of beef cattle in North America. Mannheimia haemolytica is the most common cause of BRD, but recent work suggests that Pasteurella multocida is increasing in importance. Because P multocida is a commensal inhabitant of the upper respiratory tract, it is generally considered an opportunistic pathogen. However, studies in swine indicated that there may be a limited number of strains associated with disease, suggesting that some are more virulent than others. We hypothesize that similar strain differences exist in cattle. Polymerization chain reaction (PCR) fingerprinting is effective in discriminating between isolates of P multocida, but it has not been validated in bovine isolate.s. Therefore, the purpose of this study was to compare the effectiveness of PCR fingerprinting of P multocida isolates from cases of BRD to more traditional approaches, including whole cell protein (WCP) profiles, outer membrane protein (OMP) profiles and serotyping. The information obtained through these techniques was then used to examine isolate diversity to determine if disease was primarily attributable to a limited number of strains.