Multiple virulence factors regulated by AlgU contribute to the pathogenicity of Pseudomonas savastanoi pv. glycinea in soybean

Pseudomonas savastanoi pv. glycinea (Psg) causes bacterial blight of soybean. To identify candidate virulence factors, transposon-mediated mutational analysis of Psg was carried out. We syringe-inoculated soybean leaves with Psg transposon mutants and identified 28 mutants which showed reduced virulence from 1,000 mutants screened. Next, we spray-inoculated soybean leaves with these mutants and demonstrated that the algU mutant showed significantly reduced virulence together with reduced bacterial populations in planta. Expression profiles comparison between the Psg wild-type (WT) and algU mutant in HSC broth revealed that expression of coronatine (COR)-related genes (including cmaA and corR) were down-regulated in the algU mutant compared with Psg WT. Moreover, we also showed that COR production were reduced in the algU mutant compared with WT. We also demonstrated that algD, which is related to alginate biosynthesis, showed reduced expression and biofilm formation was significantly suppressed in the algU mutant. Furthermore, hrpL also showed less expression in the algU mutant. These results indicate that AlgU plays a critical role in promoting Psg pathogenesis by regulating multiple virulence factors.

Identification of a PadR-type regulator essential for intracellular pathogenesis of Burkholderia pseudomallei

Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a disease endemic to the tropics. Melioidosis manifests in various ways ranging from acute skin lesions to pneumonia and, in rare cases, infection of the central nervous system. Bp is a facultative intracellular pathogen and it can infect various cell types. The Bp intracellular lifecycle has been partially elucidated and is highly complex. Herein, we have identified a transcriptional regulator, BP1026B_II1198, that is differentially expressed as Bp transits through host cells. A deletion mutant of BP1026B_II1198 was attenuated in RAW264.7 cell and BALB/c mouse infection. To further characterize the function of this transcriptional regulator, we endeavored to determine the regulon of BP1026B_II1198. RNA-seq analysis showed the global picture of genes regulated while ChIP-seq analysis identified two specific BP1026B_II1198 binding regions on chromosome II. We investigated the transposon mutants of these genes controlled by BP1026B_II1198 and confirmed that these genes contribute to pathogenesis in RAW264.7 murine macrophage cells. Taken together, the data presented here shed light on the regulon of BP1026B_II1198 and its role during intracellular infection and highlights an integral portion of the highly complex regulation network of Bp during host infection.

A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impact Klebsiella pneumoniae fitness

Hypervirulent K. pneumoniae (hvKp) is a distinct pathotype that causes invasive community-acquired infections in healthy individuals. Hypermucoviscosity (hmv) is a major phenotype associated with hvKp characterized by copious capsule production and poor sedimentation. Dissecting the individual functions of CPS production and hmv in hvKp has been hindered by the conflation of these two properties. Although hmv requires capsular polysaccharide (CPS) biosynthesis, other cellular factors may also be required and some fitness phenotypes ascribed to CPS may be distinctly attributed to hmv. To address this challenge, we systematically identified genes that impact capsule and hmv. We generated a condensed, ordered transposon library in hypervirulent strain KPPR1, then evaluated the CPS production and hmv phenotypes of the 3,733 transposon mutants, representing 72% of all open reading frames in the genome. We employed forward and reverse genetic screens to evaluate effects of novel and known genes on CPS biosynthesis and hmv. These screens expand our understanding of core genes that coordinate CPS biosynthesis and hmv, as well as identify central metabolism genes that distinctly impact CPS biosynthesis or hmv, specifically those related to purine metabolism, pyruvate metabolism and the TCA cycle. Six representative mutants, with varying effect on CPS biosynthesis and hmv, were evaluated for their impact on CPS thickness, serum resistance, host cell association, and fitness in a murine model of disseminating pneumonia. Altogether, these data demonstrate that hmv requires both CPS biosynthesis and other cellular factors, and that hmv and CPS may serve distinct functions during pathogenesis. The integration of hmv and CPS to the metabolic status of the cell suggests that hvKp may require certain nutrients to specifically cause deep tissue infections.

Four families of folate-independent methionine synthases

Although most organisms synthesize methionine from homocysteine and methyl folates, some have “core” methionine synthases that lack folate-binding domains and use other methyl donors. In vitro, the characterized core synthases use methylcobalamin as a methyl donor, but in vivo, they probably rely on corrinoid (vitamin B12-binding) proteins. We identified four families of core methionine synthases that are distantly related to each other (under 30% pairwise amino acid identity). From the characterized enzymes, we identified the families MesA, which is found in methanogens, and MesB, which is found in anaerobic bacteria and archaea with the Wood-Ljungdahl pathway. A third uncharacterized family, MesC, is found in anaerobic archaea that have the Wood-Ljungdahl pathway and lack known forms of methionine synthase. We predict that most members of the MesB and MesC families accept methyl groups from the iron-sulfur corrinoid protein of that pathway. The fourth family, MesD, is found only in aerobic bacteria. Using transposon mutants and complementation, we show that MesD does not require 5-methyltetrahydrofolate or cobalamin. Instead, MesD requires an uncharacterized protein family (DUF1852) and oxygen for activity.

Identification of Membrane-Bound Lytic Murein Transglycosylase A (MltA) as a Growth Factor for Francisella novicida in a Silkworm Infection Model

Francisella tularensis, the causative agent of tularemia, is transmitted by arthropod vectors within mammalian hosts. The detailed mechanisms contributing to growth and survival of Francisella within arthropod remain poorly understood. To identify novel factors supporting growth and survival of Francisella within arthropods, a transposon mutant library of F. tularensis subsp. novicida (F. novicida) was screened using an F. novicida–silkworm infection model. Among 750 transposon mutants screened, the mltA-encoding membrane-bound lytic murein transglycosylase A (MltA) was identified as a novel growth factor of F. novicida in silkworms. Silkworms infection with an mltA deletion mutant (ΔmltA) resulted in a reduction in the number of bacteria and prolonged survival. The ΔmltA strain exhibited limited intracellular growth and cytotoxicity in BmN4 silkworm ovary cells. Moreover, the ΔmltA strain induced higher expression of the antimicrobial peptide in silkworms compared to the wild-type strain. These results suggest that F. novicida MltA contributes to the survival of F. novicida in silkworms via immune suppression-related mechanisms. Intracellular growth of the ΔmltA strain was also reduced in human monocyte THP-1 cells. These results also suggest the contribution of MltA to pathogenicity in humans and utility of the F. novicida–silkworm infection model to explore Francisella infection.

BBB07 contributes to, but is not essential for, Borrelia burgdorferi infection in mice

Borrelia burgdorferi, a causative agent of Lyme disease, encodes a protein BBB07 on the genomic plasmid cp26. BBB07 was identified as a candidate integrin ligand based on the presence of an RGD tripeptide motif, which is present in a number of mammalian ligands for β1 and β3 integrins . Previous work demonstrated that BBB07 in recombinant form binds to β1 integrins and induces inflammatory responses in synovial cells in culture. Several transposon mutants in bbb07 were attenuated in an in vivo screen of the transposon library in mice. We therefore tested individual transposon mutant clones in single-strain infections in mice and found that they were attenuated in terms of ID50 but did not have significantly reduced tissue burdens in mice. Based on data presented here we conclude that BBB07 is not essential for, but does contribute to, B. burgdorferi infectivity in mice.

A Model Roseobacter, Ruegeria pomeroyi DSS-3, Employs a Diffusible Killing Mechanism To Eliminate Competitors

ABSTRACT The Roseobacter clade is a group of alphaproteobacteria that have diverse metabolic and regulatory capabilities. They are abundant in marine environments and have a substantial role in marine ecology and biogeochemistry. However, interactions between roseobacters and other bacterioplankton have not been extensively explored. In this study, we identify a killing mechanism in the model roseobacter Ruegeria pomeroyi DSS-3 by coculturing it with a group of phylogenetically diverse bacteria. The killing mechanism is diffusible and occurs when cells are grown both on surfaces and in suspension and is dependent on cell density. A screen of random transposon mutants revealed that the killing phenotype, as well as resistance to killing, require genes within an ∼8-kb putative gamma-butyrolactone synthesis gene cluster, which resembles similar pheromone-sensing systems in actinomycetes that regulate secondary metabolite production, including antimicrobials. Transcriptomics revealed the gene cluster is highly upregulated in wild-type DSS-3 compared to a nonkiller mutant when grown in liquid coculture with a roseobacter target. Our findings show that R. pomeroyi has the capability to eliminate closely and distantly related competitors, providing a mechanism to alter the community structure and function in its native habitats. IMPORTANCE Bacteria carry out critical ecological and biogeochemical processes and form the foundations of ecosystems. Identifying the factors that influence microbial community composition and the functional capabilities encoded within them is key to predicting how microbes impact an ecosystem. Because microorganisms must compete for limited space and nutrients to promote their own propagation, they have evolved diverse mechanisms to outcompete or kill competitors. However, the genes and regulatory strategies that promote such competitive abilities are largely underexplored, particularly in free-living marine bacteria. Here, genetics and omics techniques are used to investigate how a model marine bacterium is capable of quickly eliminating natural competitors in coculture. We determined that a previously uncharacterized horizontally acquired gene cluster is required for this bacterium to kill diverse competitors. This work represents an important step toward understanding the mechanisms bacterial populations can use to become dominant members in marine microbial communities.

Mycobacterium Von Willebrand factor protein MSMEG_3641 is involved in biofilm formation and intracellular survival

Aim: Mycobacterium tuberculosis in vitro biofilm is associated with the virulence and persistence capability. Our aim is to delineate factors involved in biofilms development. Materials & methods: We performed transposon mutants screen and found that mutation of MSMEG_3641, a homolog of M. tuberculosis Rv1836c, can change M. smegmatis colony morphology and biofilm. Results: MSMEG_3641 contains a vWA domain that is highly conserved among Mycobacteria. The phenotypes of MSMEG_3641 mutants include disrupted biofilm, weakened migration ability and changed colony morphology. All phenotypes might be contributed to the enhanced cell wall permeability and declined cell aggregation ability. Conclusion: To our knowledge, this is the first report concerning the mycobacteria Von Willebrand factor domain function, especially in colony morphology and biofilm development.

Four Families of Folate-independent Methionine Synthases

Although most organisms synthesize methionine from homocysteine and methyl folates, some have “core” methionine synthases that lack folate-binding domains and use other methyl donors. In vitro, the characterized core synthases use methylcobalamin as a methyl donor, but in vivo, they probably rely on corrinoid (vitamin B12-binding) proteins. We identified four families of core methionine synthases that are distantly related to each other (under 30% pairwise amino acid identity). From the characterized enzymes, we identified the families MesA, which is found in methanogens, and MesB, which is found in anaerobic bacteria and archaea with the Wood-Ljungdahl pathway. A third uncharacterized family, MesC, is found in anaerobic archaea that have the Wood-Ljungdahl pathway and lack known forms of methionine synthase. We predict that most members of the MesB and MesC families accept methyl groups from the iron-sulfur corrinoid protein of that pathway. The fourth family, MesD, is found only in aerobic bacteria. Using transposon mutants and complementation, we show that MesD does not require 5-methyltetrahydrofolate or cobalamin. Instead, MesD requires an uncharacterized protein family (DUF1852) and oxygen for activity.