Denise K. Zinniel

Isolation and Characterization of Endophytic Colonizing Bacteria from Agronomic Crops and Prairie Plants

ABSTRACT Endophytic bacteria reside within plant hosts without causing disease symptoms. In this study, 853 endophytic strains were isolated from aerial tissues of four agronomic crop species and 27 prairie plant species. We determined several phenotypic properties and found approximately equal numbers of gram-negative and gram-positive isolates. In a greenhouse study, 28 of 86 prairie plant endophytes were found to colonize their original hosts at 42 days postinoculation at levels of 3.5 to 7.7 log10 CFU/g (fresh weight). More comprehensive colonization studies were conducted with 373 corn and sorghum endophytes. In growth room studies, none of the isolates displayed pathogenicity, and 69 of the strains were recovered from corn or sorghum seedlings at levels of 8.3 log10 CFU/plant or higher. Host range greenhouse studies demonstrated that 26 of 29 endophytes were recoverable from at least one host other than corn and sorghum at levels of up to 5.8 log10 CFU/g (fresh weight). Long-range dent corn greenhouse studies and field trials with 17 wild-type strains and 14 antibiotic-resistant mutants demonstrated bacterial persistence at significant average colonization levels ranging between 3.4 and 6.1 log10 CFU/g (fresh weight) up to 78 days postinoculation. Three prairie and three agronomic endophytes exhibiting the most promising levels of colonization and an ability to persist were identified as Cellulomonas, Clavibacter, Curtobacterium, and Microbacterium isolates by 16S rRNA gene sequence, fatty acid, and carbon source utilization analyses. This study defines for the first time the endophytic nature of Microbacterium testaceum. These microorganisms may be useful for biocontrol and other applications.

Bayesian Analyses of Multiple Epistatic QTL Models for Body Weight and Body Composition in Mice

To comprehensively investigate the genetic architecture of growth and obesity, we performed Bayesian analyses of multiple epistatic quantitative trait locus (QTL) models for body weights at five ages (12 days, 3, 6, 9 and 12 weeks) and body composition traits (weights of two fat pads and five organs) in mice produced from a cross of the F1 between M16i (selected for rapid growth rate) and CAST/Ei (wild-derived strain of small and lean mice) back to M16i. Bayesian model selection revealed a temporally regulated network of multiple QTL for body weight, involving both strong main effects and epistatic effects. No QTL had strong support for both early and late growth, although overlapping combinations of main and epistatic effects were observed at adjacent ages. Most main effects and epistatic interactions had an opposite effect on early and late growth. The contribution of epistasis was more pronounced for body weights at older ages. Body composition traits were also influenced by an interacting network of multiple QTLs. Several main and epistatic effects were shared by the body composition and body weight traits, suggesting that pleiotropy plays an important role in growth and obesity.

Metabolomics Analysis Identifies d-Alanine-d-Alanine Ligase as the Primary Lethal Target of d-Cycloserine in Mycobacteria

d-Cycloserine is an effective second line antibiotic used as a last resort to treat multi (MDR)- and extensively (XDR) drug resistant strains of Mycobacterium tuberculosis . d-Cycloserine interferes with the formation of peptidoglycan biosynthesis by competitive inhibition of alanine racemase (Alr) and d-alanine-d-alanine ligase (Ddl). Although the two enzymes are known to be inhibited, the in vivo lethal target is still unknown. Our NMR metabolomics work has revealed that Ddl is the primary target of DCS, as cell growth is inhibited when the production of d-alanyl-d-alanine is halted. It is shown that inhibition of Alr may contribute indirectly by lowering the levels of d-alanine, thus allowing DCS to outcompete d-alanine for Ddl binding. The NMR data also supports the possibility of a transamination reaction to produce d-alanine from pyruvate and glutamate, thereby bypassing Alr inhibition. Furthermore, the inhibition of peptidoglycan synthesis results in a cascading effect on cellular metabolism as there is a shift toward the catabolic routes to compensate for accumulation of peptidoglycan precursors.

Genetic Resistance of Mice to Mycobacterium paratuberculosis Is Influenced by Slc11a1 at the Early but Not at the Late Stage of Infection

ABSTRACT We have recently described the development of a luminescent Mycobacterium paratuberculosis strain of bovine origin expressing the luxAB genes of Vibrio harveyi. With this luminescent isolate, fastidious and costly enumeration of CFU by plating them on agar can be replaced by easy and rapid luminometry. Here, we have reevaluated the effect of Slc11a1 (formerly Nramp1) polymorphism on susceptibility to M. paratuberculosis, using this luminometric method. A series of inbred mouse strains were infected intravenously with luminescent M. paratuberculosis S-23 and monitored for bacterial replication in spleen, liver, and lungs for 12 weeks. The results indicate that, as for Mycobacterium avium subsp. avium, innate resistance to infection is genetically controlled by Slc11a1. In BALB/c, congenic BALB.B10-H2b (BALB/c background; H-2b), C57BL/6, and beige C57BL/6bg/bg mice (all Slc11a1s), bacterial numbers in spleen and liver remained unchanged during the first 4 weeks of infection, whereas in DBA/2 and congenic BALB/c.DBA/2 (C.D2) mice (both Slc11a1r) and in (C57BL/6 × DBA/2)F1 mice (Slc11a1s/r), the bacterial numbers had decreased more than 10-fold at 4 weeks postinfection in both male and female mice. At later time points, additional differences in bacterial replication were observed between the susceptible mouse strains, particularly in the liver. Whereas bacterial numbers in the liver gradually decreased more than 100-fold in C57BL/6 mice between week 4 and week 12, bacterial numbers were stable in livers from BALB/c and beige C57BL/6bg/bg mice during this period. Mycobacterium-specific gamma interferon responses developed earlier and to a higher magnitude in C57BL/6 mice than in BALB/c mice and were lowest in resistant C.D2 mice.

Development of luminescent Mycobacterium avium subsp. paratuberculosis for rapid screening of vaccine candidates in mice.

ABSTRACT Mycobacterium avium subsp. paratuberculosis is a slowly growing mycobacterial species, requiring 6 to 8 weeks of culture before colonies can be counted visually. Here, we describe the development of luminescent M. avium subsp. paratuberculosis expressing luxAB genes of Vibrio harveyi and its use for vaccine testing in an experimental mouse model, replacing fastidious CFU counting by rapid luminometry.

Immunogenicity and Reactivity of Novel Mycobacterium avium subsp. paratuberculosis PPE MAP1152 and Conserved MAP1156 Proteins with Sera from Experimentally and Naturally Infected Animals

ABSTRACT Mycobacterium avium subsp. paratuberculosis causes Johnes disease (JD) in ruminants. Development of genetic tools and completion of the M. avium subsp. paratuberculosis genome sequencing project have expanded the opportunities for antigen discovery. In this study, we determined the seroreactivities of two proteins encoded at the 5′ and 3′ regions of the MAP1152-MAP1156 gene cluster. MAP1152 encodes a PPE protein, and MAP1156 encodes a diacylglycerol acyltransferase involved in triglyceride metabolism and classified in the uncharacterized protein family UPF0089. Recombinant MAP proteins were overproduced and purified from Escherichia coli as maltose-binding protein (MBP) fusions. Immunoblotting analysis indicated that both MAP1152 and MAP1156 displayed reactivity against sera of mice and rabbits immunized with live M. avium subsp. paratuberculosis cells and against samples from naturally infected cattle. In immunoblot assays, MAP1156 yielded a stronger positive signal than MAP1152 against sera from cattle with JD. An enzyme-linked immunosorbent assay for the recombinant proteins was developed and used to test preclassified positive and negative serum samples from naturally infected and noninfected cattle. Samples, with one exception, displayed no seroreactivity against the MBP-LacZ fusion protein (P > 0.05), the negative-control antigen. MAP1152 displayed seroreactivity against all positive sera but no seroreactivity to the negative sera (P < 0.01). MAP1156 displayed stronger and more variable reactivity than MAP1152, but significant differences were observed between noninfected and infected cattle (P < 0.05). Otherwise, degrees of reactivity followed the same trend as the positive reference antigen. In conclusion, both proteins are immunogenic in mice and rabbits, and M. avium subsp. paratuberculosis-infected cattle mount a humoral response to both MAP1152 and MAP1156 cross-reactive epitopes. These findings have potential applications to diagnostics, vaccine production, and elucidation of the immunopathogenesis of JD.

Impairment of D-alanine biosynthesis in Mycobacterium smegmatis determines decreased intracellular survival in human macrophages

d-Alanine is a structural component of mycobacterial peptidoglycan. The primary route of d-alanine biosynthesis in eubacteria is the enantiomeric conversion from l-alanine, a reaction catalysed by d-alanine racemase (Alr). Mycobacterium smegmatis alr insertion mutants are not dependent on d-alanine for growth and display a metabolic pattern consistent with an alternative pathway for d-alanine biosynthesis. In this study, we demonstrate that the M. smegmatis alr insertion mutant TAM23 can synthesize d-alanine at lower levels than the parental strain. The insertional inactivation of the alr gene also decreases the intracellular survival of mutant strains within primary human monocyte-derived macrophages. By complementation studies, we confirmed that the impairment of alr gene function is responsible for this reduced survival. Inhibition of superoxide anion and nitric oxide formation in macrophages suppresses the differential survival. In contrast, for bacteria grown in broth, both strains had approximately the same susceptibility to hydrogen peroxide, acidified sodium nitrite, low pH and polymyxin B. In contrast, TAM23 exhibited increased resistance to lysozyme. d-Alanine supplementation considerably increased TAM23 viability in nutritionally deficient media and within macrophages. These results suggest that nutrient deprivation in phagocytic cells combined with killing mediated by reactive intermediates underlies the decreased survival of alr mutants. This knowledge may be valuable in the construction of mycobacterial auxotrophic vaccine candidates.

Generation and screening of a comprehensive Mycobacterium avium subsp. paratuberculosis transposon mutant bank

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johnes Disease in ruminants. This enteritis has significant economic impact and worldwide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay development. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P > 95%) was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped upstream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated.

Molecular analysis of the Mycobacterium tuberculosis lux-like mel2 operon

Using a high throughput genetic strategy, designated Random Inducible Controlled Expression (RICE), we identified the six gene mel2 locus in Mtb and M. marinum. Interestingly, three of the genes present in mel2 have similarities to bioluminescence genes. Similar to other bacterial bioluminescence systems, mel2 facilitates detoxification of reactive oxygen species (ROS). Through the use of thin layer chromatography (TLC) we demonstrate enhanced production of the cell wall virulence lipid, pthiocerol dimycoserosate (PDIM), in a Mtb mel2 mutant relative to the wild type strain in the presence of both H2O2 and diamide oxidative stresses. Furthermore, propionate toxicity assays revealed increased accumulation of triacylglycerol (TAG) in the mel2 mutant relative to wild type. These observations provide the first evidence that mel2 plays a critical role in Mtb lipid biosynthesis.

Assessment of Metabolic Changes in Mycobacterium smegmatis Wild-Type and alr Mutant Strains: Evidence of a New Pathway of d-Alanine Biosynthesis

In mycobacteria, d-alanine is an essential precursor for peptidoglycan biosynthesis. The only confirmed enzymatic pathway to form d-alanine is through the racemization of l-alanine by alanine racemase (Alr, EC 5.1.1.1). Nevertheless, the essentiality of Alr in Mycobacterium tuberculosis and Mycobacterium smegmatis for cell survivability in the absence of d-alanine has been a point of controversy with contradictory results reported in the literature. To address this issue, we examined the effects of alr inactivation on the cellular metabolism of M. smegmatis. The M. smegmatis alr insertion mutant TAM23 exhibited essentially identical growth to wild-type mc2155 in the absence of d-alanine. NMR metabolomics revealed drastically distinct phenotypes between mc2155 and TAM23. A metabolic switch was observed for TAM23 as a function of supplemented d-alanine. In the absence of d-alanine, the metabolic response directed carbon through an unidentified transaminase to provide the essential d-alanine required for survival. The process is reversed when d-alanine is available, in which the d-alanine is directed to peptidoglycan biosynthesis. Our results provide further support for the hypothesis that Alr is not an essential function of M. smegmatis and that specific Alr inhibitors will have no bactericidal action.