aro mutations in Salmonella enterica cause defects in cell wall and outer membrane integrity

In this study we characterized aro mutants of Salmonella enterica serovars Enteritidis and Typhimurium, which are frequently used as live oral vaccines. We found that the aroA, aroD, and aroC mutants were sensitive to blood serum, albumen, EDTA, and ovotransferrin, and this defect could be complemented by an appropriate aro gene cloned in a plasmid. Subsequent microarray analysis of gene expression in the aroD mutant in serovar Typhimurium indicated that the reason for this sensitivity might be the upregulation of murA. To confirm this, we artificially overexpressed murA from a multicopy plasmid, and this overexpression caused sensitivity of the strain to albumen and EDTA but not to serum and ovotransferrin. We concluded that attenuation of aro mutants is caused not only by their inability to synthesize aromatic metabolites but also by their defect in cell wall and outer membrane functions associated with decreased resistance to components of innate immune response.     

福氏2a志贺氏菌△aroA突变减毒株的构建

志贺氏菌芳香族氨基酸合成酶基因缺陷能够使菌体明显减毒,并有可能成为新一代痢疾疫苗．用ＰＣＲ技术从野生型福氏２ａ志贺氏菌２４５７Ｔ中克隆出ａｒｏＡ基因,在体外进行精确的缺失突变,并通过体内同源重组,构建成△ａｒｏＡ突变体ＲＳ４２６．实验结果表明,这种突变体仍保持了侵袭能力和保护性Ｏ抗原的表达,但其毒力已明显降低,不能产生豚鼠角结膜炎,小鼠半数致死量明显提高．免疫保护试验显示,ＲＳ４２６可在小鼠中产生对福氏２ａ野生菌１００％的保护作用．     

Genes aroA and serC of Salmonella typhimurium constitute an operon.

Genetic analysis of aroA554::Tn10 derivatives of two mouse-virulent Salmonella typhimurium strains, "FIRN" and "WRAY," and of a nonreverting derivative of each constructed for use as a live vaccine, showed the site of the insertion among mapped aroA point mutants. The WRAY live-vaccine strain gave no aro+ recombinants in crosses with aroA point mutations to one side of the insertion, indicating a deletion from Tn10 through the sites of these point mutations. The FIRN live-vaccine strain gave wild-type recombinants with all tested point mutants; it probably has a deletion or inversion extending from Tn10 into aroA but not as far as the nearest point mutation. Some tetracycline-sensitive mutants of aroA554::Tn10 strains required serine and pyridoxine, indicating loss of serC function, and some that were found to be SerC- did not produce gas from glucose, indicating a loss of pfl function. These results show the gene order pfl-serC-aroA, as in Escherichia coli. Ampicillin enrichment applied to pools of tetracycline-sensitive mutants of strains with Tn10 insertions near aroA (i.e., zbj::Tn10 strains) yielded Aro- SerC- Pfl-, Aro- SerC+ Pfl+, and Aro- SerC- Pfl+ mutants but none which were Aro+ SerC-. All of the mutants are explicable by deletions or inversions extending clockwise from zbj::Tn10 into or through an operon comprising serC (promoter-proximal) and aroA. Such an operon was also shown by the identification of two Tn10 insertions causing phenotype Aro- SerC-, each able to revert to Aro+ SerC+ by precise excision. serC corresponds to the open reading frame promoter-proximal to aroA that was identified elsewhere by base sequencing of a cloned aroA segment of S. typhimurium (Comai et al., Science 221:370-371, 1983). Both serine and chorismate are precursors of enterochelin; this may be why serC and aroA are in a single operon.     

Identification of exopolysaccharide-deficient mutants of Mycoplasma pulmonis

The presence of capsular exopolysaccharide (EPS) in Mollicutes has been inferred from electron micrographs for over 50 years without conclusive data to support the production of complex carbohydrates by the organism. Mycoplasma pulmonis binds the lectin Griffonia simplicifolia I (GS-I), which is specific for terminal β-linked galactose residues. Mutants that failed to produce the EPS bound by GS-I were isolated from a transposon library. All of the mutants had the transposon located in open reading frame MYPU_7410 or MYPU_7420. These overlapping genes are predicted to code for a heterodimeric pair of ABC transporter permeases and may code for part of a new pathway for synthesis of EPS. Analysis by lectin-affinity chromatography in conjunction with gas chromatography demonstrated that the wild-type mycoplasma produced an EPS (EPS-I) composed of equimolar amounts of glucose and galactose that was lacking in the mutants. Phenotypic analysis revealed that the mutants had an increased propensity to form a biofilm on glass surfaces, colonized mouse lung and trachea efficiently, but had a decreased association with the A549 lung cell line. Confounding the interpretation of these results is the observation that the mutants missing EPS-I had an eightfold overproduction of an apparent second EPS (EPS-II) containing N -acetylglucosamine.     

Novel ATP-driven pathway of glycolipid export involving TolC protein

Upon depletion of combined nitrogen, N(2)-fixing heterocysts are formed from vegetative cells in the case of the filamentous cyanobacterium Anabaena sp. strain PCC 7120. A heterocyst-specific layer composed of glycolipids (heterocyst envelope glycolipids (HGLs)) that functions as an O(2) diffusion barrier is deposited over the heterocyst outer membrane and is surrounded by an outermost heterocyst polysaccharide envelope. Mutations in any gene of the devBCA operon or tolC result in the absence of the HGL layer, preventing growth on N(2) used as the sole nitrogen source. However, those mutants do not have impaired HGL synthesis. In this study, we show that DevBCA and TolC form an ATP-driven efflux pump required for the export of HGLs across the Gram-negative cell wall. By performing protein-protein interaction studies (in vivo formaldehyde cross-linking, surface plasmon resonance, and isothermal titration calorimetry), we determined the kinetics and stoichiometric relations for the transport process. For sufficient glycolipid export, the membrane fusion protein DevB had to be in a hexameric form to connect the inner membrane factor DevC and the outer membrane factor TolC. A mutation that impaired the ability of DevB to form a hexameric arrangement abolished the ability of DevC to recognize its substrate. The physiological relevance of a hexameric DevB is shown in complementation studies. We provide insights into a novel pathway of glycolipid export across the Gram-negative cell wall.     

Proteomic studies highlight outer‐membrane proteins related to biofilm development in the marine bacterium Pseudoalteromonas sp. D41

Bacterial biofilm development is conditioned by complex processes involving bacterial attachment to surfaces, growth, mobility, and exoproduct production. The marine bacterium Pseudoalteromonas sp. strain D41 is able to attach strongly onto a wide variety of substrates, which promotes subsequent biofilm development. Study of the outer‐membrane and total soluble proteomes showed ten spots with significant intensity variations when this bacterium was grown in biofilm compared to planktonic cultures. MS/MS de novo sequencing analysis allowed the identification of four outer‐membrane proteins of particular interest since they were strongly induced in biofilms. These proteins are homologous to a TonB‐dependent receptor (TBDR), to the OmpW and OmpA porins, and to a type IV pilus biogenesis protein (PilF). Gene expression assays by quantitative RT‐PCR showed that the four corresponding genes were upregulated during biofilm development on hydrophobic and hydrophilic surfaces. The Pseudomonas aeruginosa mutants unable to produce any of the OmpW, OmpA, and PilF homologues yielded biofilms with lower biovolumes and altered architectures, confirming the involvement of these proteins in the biofilm formation process. Our results indicate that Pseudoalteromonas sp. D41 shares biofilm formation mechanisms with human pathogenic bacteria, but also relies on TBDR, which might be more specific to the marine environment.     

Spent media from cultures of environmental isolates of Escherichia coli can suppress the deficiency of biofilm formation under anoxic conditions of laboratory E. coli strains

The prevailing lifestyle of bacteria is sessile and they attach to surfaces in structures known as biofilms. In Escherichia coli, as in many other bacteria, biofilms are formed at the air-liquid interface, suggesting that oxygen has a critical role in the biofilm formation process. It has been reported that anaerobically growing E. coli laboratory strains are unable to form biofilms even after 96 h of incubation on Luria Bertani (LB) medium. After analyzing 22,000 transposon-induced and 26,000 chemically-induced mutants we failed to isolate an E. coli laboratory strain with the ability to form biofilm under anaerobic growth conditions. Notably, seven strains from a collection of E. coli isolated from different hosts and the environment had the ability to form biofilm in the absence of oxygen. Interestingly, spent medium from cultures of one strain, Souza298, can promote biofilm formation of E. coli laboratory strains growing under anaerobic conditions. Our results led us to propose that laboratory E. coli strains do not release (or synthesize) a molecule needed for biofilm formation under anoxic conditions but that they bear all the required machinery needed for this process.     

Mycoplasma pulmonis Vsa proteins and polysaccharide modulate adherence to pulmonary epithelial cells

The Mycoplasma pulmonis Vsa proteins are a family of size- and phase-variable lipoproteins that shield the mycoplasmas from complement and modulate attachment to abiotic surfaces. Mycoplasmas producing a long Vsa protein hemadsorb poorly and yet are proficient at colonizing rats and mice. The effect of the length of the Vsa protein on the attachment of mycoplasmas to epithelial cells has not been previously explored. We find that independent of Vsa isotype, mycoplasmas producing a long Vsa protein with many tandem repeats adhere poorly to murine MLE-12 cells compared with mycoplasmas producing a short Vsa. We also find that mutants lacking the EPS-I polysaccharide of M.?pulmonis exhibited decreased adherence to MLE-12 cells, even though it has been shown previously that such mutants have an enhanced ability to form a biofilm.     

Outer membrane protein a and other polypeptides regulate capsular polysaccharide synthesis in E. coli K-12.

Summary capR (lon) mutants of Escherichia coli K-12 are mucoid on minimal agar because they produce large quantities of capsular polysaccharide. When such mutants are transformed to tetracycline resistance by plasmid pMC44, a hybrid plasmid that contains a 2 megadalton (Mdal) endonuclease EcoR1 fragment of E. coli K-12 DNA joined to the cloning vehicle-pSC101, capsular polysaccharide synthesis is inhibited and the transformed colonies exhibit a nonmucoid phenotype. Re-cloning of the 2 Mdal EcoR1 fragment onto plasmid pHA105, a min-colE1 plasmid, yielded plasmid pFM100 which also inhibited capsular polysaccharide synthesis in the capR mutants. A comparison of the polypeptides specified by both plasmids pFM100 and pMC44 in minicells demonstrated that seven polypeptide bands were specified by the 2 Mdal DNA, one of which was previously demonstrated to be outer membrane protein a; also known as 3b or M2 (40 kilodaltons, Kdal). Plasmid mutants no longer repressing capsular polysaccharide synthesis were either unable to specify the 40 K dal outer membrane protein a or were deficient in synthesis of 25 K dal and 14.5 K dal polypeptides specified by the 2 Mdal DNA fragment. Studies with a minicell-producing strain that also contained a capR mutation indicated that the capR gene product regulated processing of at least one normal protein, the precursor of outer membrane protein a.     

The effect of mutations in the synthesis of lipopolysaccharides and calcofluor-binding polysaccharides on biofilm formation by Azospirillum brasilense

The thickness and antigenic properties of biofilms produced by Azospirillum brasilense Sp245 and its mutants deficient in the synthesis of lipopolysaccharides (Lps) and calcofluor-binding polysaccharides (CBPS) at the interface between water and hydrophilic or hydrophobic solid surfaces were compared. The mutants deficient in acidic LpsI synthesis produce thicker biofilms on hydrophilic surfaces. Biofilms produced on hydrophobic surfaces by bacteria that are unable to synthesize CBPS are less pronounced. Defects in CBPS production in Azospirillum mutants with impaired flagellar motility can cause adverse effects on the cell ability to attach to hydrophobic and hydrophilic surfaces. The loss of the neutral LpsII antigen by the mutants capable of producing CBPS does not affect their behavior on hydrophobic surfaces, which is probably due to the compensatory increase in the total polysaccharide production. The fundamental change in the Lps structure correlates with the activation of biofilm formation by the relevant mutants on hydrophilic and hydrophobic surfaces.     

The Fusobacterium nucleatum outer membrane protein RadD is an arginine-inhibitable adhesin required for inter-species adherence and the structured architecture of multispecies biofilm

A defining characteristic of the suspected periodontal pathogen Fusobacterium nucleatum is its ability to adhere to a plethora of oral bacteria. This distinguishing feature is suggested to play an important role in oral biofilm formation and pathogenesis, with fusobacteria proposed to serve as central 'bridging organisms' in the architecture of the oral biofilm bringing together species which would not interact otherwise. Previous studies indicate that these bacterial interactions are mediated by galactose- or arginine-inhibitable adhesins although genetic evidence for the role and nature of these proposed adhesins remains elusive. To characterize these adhesins at the molecular level, the genetically transformable F. nucleatum strain ATCC 23726 was screened for adherence properties, and arginine-inhibitable adhesion was evident, while galactose-inhibitable adhesion was not detected. Six potential arginine-binding proteins were isolated from the membrane fraction of F. nucleatum ATCC 23726 and identified via mass spectroscopy as members of the outer membrane family of proteins in F. nucleatum . Inactivation of the genes encoding these six candidates for arginine-inhibitable adhesion and two additional homologues revealed that only a mutant derivative carrying an insertion in Fn1526 (now designated as radD ) demonstrated significantly decreased co-aggregation with representatives of the Gram-positive 'early oral colonizers'. Lack of the 350 kDa outer membrane protein encoded by radD resulted in the failure to form the extensive structured biofilm observed with the parent strain when grown in the presence of Streptococcus sanguinis ATCC 10556. These findings indicate that radD is responsible for arginine-inhibitable adherence of F. nucleatum and provides definitive molecular evidence that F. nucleatum adhesins play a vital role in inter-species adherence and multispecies biofilm formation.     

The Activities of Adhesion and Biofilm Formation by <Emphasis Type="Italic">Candida tropicalis</Emphasis> Clinical Isolates Display Significant Correlation with Its Multilocus Sequence Typing

Adhesion and biofilm formation, which can occur on abiotic and biotic surfaces, are key components in Candida pathogenicity. The aims of this study were to infer about the C. tropicalis clinical isolates ability to adhere and form biofilm on abiotic and biotic surfaces and to correlate that with the multilocus sequence typing and other virulence factors. Adhesion and biofilm formation were measured in 68 C. tropicalis isolates from 3 hospitals in China on abiotic (polystyrene) and biotic (human urinary bladder epithelial cell) surfaces by crystal violet assay and 2,3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide reduction assay. In our study, almost all C. tropicalis isolates could adhere and produce biofilm on abiotic and biotic surfaces in a strain-dependent manner. The isolates from blood showed relatively lower adhesion and biofilm capacity on polystyrene surface, but had strong secreted aspartyl proteinase activity. Moreover, significant differences were found among MLST groups for adhesion and biofilm capacity. C. tropicalis in multilocus sequence typing group5 and group6 showed high adhesion and biofilm, while isolates in group1 exhibited low adhesion and biofilm formation. Overall, it is important to note that C. tropicalis isolates adhere to and produce biofilm on abiotic and biotic surfaces with strain specificity. These data will play an important role in subsequent research on the pathogenesis of C. tropicalis.     

Pleiotropic Menaquinone-Deficient Mutant of Bacillus subtilis

A multiple aromatic amino acid auxotroph of Bacillus subtilis 168 has been isolated which is unable to synthesize menaquinone-7 (MK-7) unless supplied with shikimic acid (SHK). The mutant, RB163, was isolated by selecting for resistance to low levels (1.5 mug/ml) of kanamycin. Enzymatic and genetic analyses show that the strain is an aroD mutant lacking 5-dehydroshikimate reductase. Under growth conditions in which its MK-7 deficiency is expressed, RB163 is deficient in cytochromes a, b, and c, exhibits low growth yields, and does not sporulate. Genetic analysis indicates that this pleiotropic phenotype is the result of a single genetic event. All phenotypic characteristics are reversible when the mutant is grown under conditions such that MK is synthesized. Comparison of strain RB163 with other aro mutants blocked before SHK ("early-aro" mutants) reveals interesting differences. Most early-aro mutants are cytochrome- and MK-sufficient, sporogenous, and sensitive to kanamycin when grown in the absence of SHK. However, in addition to strain RB163, two other aro mutants were found to show the pleiotropic phenotype. These three mutants have in common, and differ from other early-aro strains in, the inability to synthesize MK. It is suggested that the phenotypically wild-type aro mutants are bradytrophic, allowing enough substrate flow through the common aromatic pathway to satisfy the MK requirement. The pleiotropic mutants are thought to be completely blocked in the common pathway, thus accounting for their inability to synthesize MK.     

The characterization of functions involved in the establishment and maturation of Klebsiella pneumoniae in vitro biofilm reveals dual roles for surface exopolysaccharides

The ability to form biofilm is seen as an increasingly important colonization strategy among both pathogenic and environmental Klebsiella pneumoniae strains. The aim of the present study was to identify abiotic surface colonization factors of K. pneumoniae using different models at different phases of biofilm development. A 2200 K. pneumoniae mutant library previously obtained by signature-tagged mutagenesis was screened in static and dynamic culture models to detect clones impaired at early and/or mature stages of biofilm formation. A total of 28 mutants were affected during late phases of biofilm formation, whereas 16 mutants displayed early adhesion defect. These mutants corresponded to genes involved in potential cellular and DNA metabolism pathways and to membrane transport functions. Eight mutants were deficient in capsule or LPS production. Gene disruption and microscopic analyses showed that LPS is involved in initial adhesion on both glass and polyvinyl-chloride and the capsule required for the appropriate initial coverage of substratum and the construction of mature biofilm architecture. These results give new insight into the bacterial factors sequentially associated with the ability to colonize an abiotic surface and reveal the dual roles played by surface exopolysaccharides during K. pneumoniae biofilm formation.     

Role for Rhizobium rhizogenes K84 cell envelope polysaccharides in surface interactions

Rhizobium rhizogenes strain K84 is a commercial biocontrol agent used worldwide to control crown gall disease. The organism binds tightly to polypropylene substrate and efficiently colonizes root surfaces as complex, multilayered biofilms. A genetic screen identified two mutants in which these surface interactions were affected. One of these mutants failed to attach and form biofilms on the abiotic surface although, interestingly, it exhibited normal biofilm formation on the biological root tip surface. This mutant is disrupted in a wcbD ortholog gene, which is part of a large locus predicted to encode functions for the biosynthesis and export of a group II capsular polysaccharide (CPS). Expression of a functional copy of wcbD in the mutant background restored the ability of the bacteria to attach and form normal biofilms on the abiotic surface. The second identified mutant attached and formed visibly denser biofilms on both abiotic and root tip surfaces. This mutant is disrupted in the rkpK gene, which is predicted to encode a UDP-glucose 6-dehydrogenase required for O-antigen lipopolysaccharide (LPS) and K-antigen capsular polysaccharide (KPS) biosynthesis in rhizobia. The rkpK mutant from strain K84 was deficient in O-antigen synthesis and exclusively produced rough LPS. We also show that strain K84 does not synthesize the KPS typical of some other rhizobia strains. In addition, we identified a putative type II CPS, distinct from KPS, that mediates cell-surface interactions, and we show that O antigen of strain K84 is necessary for normal cell-cell interactions in the biofilms.     

Identification and characterization of precursors in the biosynthesis of the K88ab fimbria of Escherichia coli

Four genes encoding for polypeptides with apparent molecular weights of 17,000, 26,000 (the fimbrial subunit), 27,000, and 81,000 have been implicated in the biosynthesis of the K88ab fimbria (Mooi et al., J. Bacteriol. 150:512-521, 1982). Escherichia coli mutants with defects in these genes were examined for the presence of fimbrial precursors. An analysis of these mutants revealed that fimbrial subunits accumulated transiently in the periplasmic space before being translocated across the outer membrane. The 81,000-dalton (d) polypeptide is probably involved in translocating fimbrial subunits across the outer membrane, because in the absence of this polypeptide the fimbrial subunits remained in the periplasmic space, where they were found to be associated with the 17,000- and 27,000-d polypeptides. In mutants with a deletion in the gene for the 27,000-d polypeptide, fimbrial precursors were not detected, because the fimbrial subunits were degraded. The 27,000-d polypeptide might be involved in stabilizing a conformation of the fimbrial subunit required to translocate it across the outer membrane. In the absence of the 17,000-d polypeptide, most fimbrial subunits were found in the periplasmic space associated with the 27,000-d polypeptide. However, small amounts of subunits were also translocated across the outer membrane. These extracellular subunits did not adhere to brushborders, suggesting that fimbrial subunits must be modified by the 17,000-d polypeptide to be assembled into functional fimbriae. A model for the biosynthesis of the K88ab fimbria is proposed.     

Phosphorylcholine expression by nontypeable Haemophilus influenzae correlates with maturation of biofilm communities in vitro and in vivo

Nontypeable Haemophilus influenzae (NTHI) causes chronic infections that feature the formation of biofilm communities. NTHI variants within biofilms have on their surfaces lipooligosaccharides containing sialic acid (NeuAc) and phosphorylcholine (PCho). Our work showed that NeuAc promotes biofilm formation, but we observed no defect in the initial stages of biofilm formation for mutants lacking PCho. In this study, we asked if alterations in NTHI PCho content affect later stages of biofilm maturation. Biofilm communities were compared for NTHI 2019 and isogenic mutants that either lacked PCho (NTHI 2019 licD) or were constitutively locked in the PCho-positive phase (NTHI 2019 lic^ON). Transformants expressing green fluorescent protein were cultured in continuous-flow biofilms and analyzed by confocal laser scanning microscopy. COMSTAT was used to quantify different biofilm parameters. PCho expression correlated significantly with increased biofilm thickness, surface coverage, and total biomass, as well as with a decrease in biofilm roughness. Comparable results were obtained by scanning electron microscopy. Analysis of thin sections of biofilms by transmission electron microscopy revealed shedding of outer membrane vesicles by NTHI bacteria within biofilms and staining of matrix material with ruthenium red in biofilms formed by NTHI 2019 lic^ON. The biofilms of all three strains were comparable in viability, the presence of extracellular DNA, and the presence of sialylated moieties on or between bacteria. In vivo infection studies using the chinchilla model of otitis media showed a direct correlation between PCho expression and biofilm formation within the middle-ear chamber and an inverse relationship between PCho and persistence in the planktonic phase in middle-ear effusions. Collectively, these data show that PCho correlates with, and may promote, the maturation of NTHI biofilms. Further, this structure may be disadvantageous in the planktonic phase.