Indole can act as an extracellular signal to regulate biofilm formation of Escherichia coli and other indole-producing bacteria

We demonstrated previously that genetic inactivation of tryptophanase is responsible for a dramatic decrease in biofilm formation in the laboratory strain Escherichia coli S17-1. In the present study, we tested whether the biochemical inhibition of tryptophanase, with the competitive inhibitor oxindolyl-L-alanine, could affect polystyrene colonization by E. coli and other indole-producing bacteria. Oxindolyl-L-alanine inhibits, in a dose-dependent manner, indole production and biofilm formation by strain S17-1 grown in Luria-Bertani (LB) medium. Supplementation with indole at physiologically relevant concentrations restores biofilm formation by strain S17-1 in the presence of oxindolyl-L-alanine and by mutant strain E. coli 3714 (S17-1 tnaA::Tn5) in LB medium. Oxindolyl-L-alanine also inhibits the adherence of S17-1 cells to polystyrene for a 3-h incubation time, but mutant strain 3714 cells are unaffected. At 0.5 mg/mL, oxindolyl-L-alanine exhibits inhibitory activity against biofilm formation in LB medium and in synthetic urine for several clinical isolates of E. coli, Klebsiella oxytoca, Citrobacter koseri, Providencia stuartii, and Morganella morganii but has no affect on indole-negative Klebsiella pneumoniae strains. In conclusion, these data suggest that indole, produced by the action of tryptophanase, is involved in polystyrene colonization by several indole-producing bacterial species. Indole may act as a signalling molecule to regulate the expression of adhesion and biofilm-promoting factors.     

Different, overlapping mechanisms for colonization of abiotic and plant surfaces by Pseudomonas putida

Mechanisms governing biofilm formation have generated considerable interest in recent years, yet comparative analyses of processes for bacterial establishment on abiotic and biotic surfaces are still limited. In this report we have expanded previous information on the genetic determinants required for colonization of plant surfaces by Pseudomonas putida populations and analyzed their correlation with biofilm formation processes on abiotic surfaces. Insertional mutations affecting flagellar genes or the synthesis and transport of the large adhesin LapA lead to decreased adhesion to seeds and biofilm formation on abiotic surfaces. The latter also causes reduced fitness in the rhizosphere. Decreased seed adhesion and altered biofilm formation kinetics are observed in mutants affected in heme biosynthesis and a gene that might participate in oxidative stress responses, whereas a mutant in a gene involved in cytochrome oxidase assembly is affected in the bacterium-plant interaction but not in bacterial establishment on abiotic surfaces. Finally, a mutant altered in lipopolysaccharide biosynthesis is impaired in seed and root colonization but seems to initiate attachment to plastic faster than the wild type. This variety of phenotypes reflects the complexity of bacterial adaptation to sessile life, and the partial overlap between mechanisms leading to biofilm formation on abiotic and biotic surfaces.     

Effect of cysteine desulfhydrase gene disruption on L-cysteine overproduction in Escherichia coli

In Escherichia coli, the enzyme called cysteine desulfhydrase (CD), which is responsible for L-cysteine degradation, was investigated by native-PAGE and CD activity staining of crude cell extracts. Analyses with gene-disrupted mutants showed that CD activity resulted from two enzymes: tryptophanase (TNase) encoded by tnaA and cystathionine beta-lyase (CBL) encoded by metC. It was also found that TNase synthesis was induced by the presence of L-cysteine. The tnaA and metC mutants transformed with the plasmid containing the gene for feedback-insensitive serine acetyltransferase exhibited higher L-cysteine productivity than the wild-type strain carrying the same plasmid. These results indicated that TNase and CBL did act on L-cysteine degradation in E. coli cells.     

UpaG, a new member of the trimeric autotransporter family of adhesins in uropathogenic Escherichia coli

The ability of Escherichia coli to colonize both intestinal and extraintestinal sites is driven by the presence of specific virulence factors, among which are the autotransporter (AT) proteins. Members of the trimeric AT adhesin family are important virulence factors for several gram-negative pathogens and mediate adherence to eukaryotic cells and extracellular matrix (ECM) proteins. In this study, we characterized a new trimeric AT adhesin (UpaG) from uropathogenic E. coli (UPEC). Molecular analysis of UpaG revealed that it is translocated to the cell surface and adopts a multimeric conformation. We demonstrated that UpaG is able to promote cell aggregation and biofilm formation on abiotic surfaces in CFT073 and various UPEC strains. In addition, UpaG expression resulted in the adhesion of CFT073 to human bladder epithelial cells, with specific affinity to fibronectin and laminin. Prevalence analysis revealed that upaG is strongly associated with E. coli strains from the B2 and D phylogenetic groups, while deletion of upaG had no significant effect on the ability of CFT073 to colonize the mouse urinary tract. Thus, UpaG is a novel trimeric AT adhesin from E. coli that mediates aggregation, biofilm formation, and adhesion to various ECM proteins.     

The flhDC gene affects motility and biofilm formation in Yersinia pseudotuberculosis

The flagella master regulatory gene flhDC of Yersinia pseudotuberculosis serotype III (YPIII) was mu- tated by deleting the middle region and replaced by a tetracycline resistant gene, and the subsequent mutant strain named YPIII?flhDC was obtained. Swimming assay showed that the swimming motility of the mutant strain was completely abolished. The promoter region of the flagella second-class regula- tory gene fliA was fused with the lux box, and was conjugated with the mutant and the parent strains respectively for the first cross. LUCY assay result demonstrated that flhDC regulated the expression of fliA in YPIII as reported in E. coli. Biofilm formation of the mutant strain on abiotic and biotic surfaces was observed and quantified. The results showed that mutation of flhDC decreased biofilm formation on both abiotic and biotic surfaces, and abated the infection on Caenorhabdtis elegans. Our results suggest that mutation of the flagella master regulatory gene flhDC not only abolished the swimming motility, but also affected biofilm formation of YPIII on different surfaces. The new function of flhDC identified in this study provides a novel viewpoint for the control of bacterial biofilm formation.     

Nucleotide sequence of the structural gene for tryptophanase of Escherichia coli K-12.

The tryptophanase structural gene, tnaA, of Escherichia coli K-12 was cloned and sequenced. The size, amino acid composition, and sequence of the protein predicted from the nucleotide sequence agree with protein structure data previously acquired by others for the tryptophanase of E. coli B. Physiological data indicated that the region controlling expression of tnaA was present in the cloned segment. Sequence data suggested that a second structural gene of unknown function was located distal to tnaA and may be in the same operon. The pattern of codon usage in tnaA was intermediate between codon usage in four of the ribosomal protein structural genes and the structural genes for three of the tryptophan biosynthetic proteins.     

L-Tryptophan prevents Escherichia coli biofilm formation and triggers biofilm degradation

The effect of deletion of trp operon and tna operon on the Escherichia coli biofilm formation was investigated in order to elucidate the role of L-tryptophan metabolism in biofilm formation. trp operon deletion mutants ΔtrpC, ΔtrpD and ΔtrpE deficient in L-tryptophan biosynthesis showed higher biofilm formation. In addition, ΔtnaC with increased L-tryptophan degradation activity showed higher biofilm formation. On the contrary, ΔtnaA deletion mutant which lost L-tryptophan degradation activity showed low biofilm formation. From these results, it was suggested that decrease of intracellular L-tryptophan level induced biofilm formation and increase of L-tryptophan repressed biofilm formation. So the effect of the addition of L-tryptophan to the medium on the E. coli biofilm formation was investigated. L-Tryptophan addition at starting culture decreased biofilm formation and furthermore L-tryptophan addition after 16 h culture induced the degradation of preformed biofilm. From the above results, it was suggested that maintenance of high intracellular L-tryptophan concentration prevents E. coli biofilm formation and elevation of intracellular L-tryptophan concentration triggers degradation of matured biofilm.     

YcfR (BhsA) influences Escherichia coli biofilm formation through stress response and surface hydrophobicity

DNA microarrays revealed that expression of ycfR, which encodes a putative outer membrane protein, is significantly induced in Escherichia coli biofilms and is also induced by several stress conditions. We show that deletion of ycfR increased biofilm formation fivefold in the presence of glucose; the glucose effect was corroborated by showing binding of the cyclic AMP receptor protein to the ycfR promoter. It appears that YcfR is a multiple stress resistance protein, since deleting ycfR also rendered the cell more sensitive to acid, heat treatment, hydrogen peroxide, and cadmium. Increased biofilm formation through YcfR due to stress appears to be the result of decreasing indole synthesis, since a mutation in the tnaA gene encoding tryptophanase prevented enhanced biofilm formation upon stress and adding indole prevented enhanced biofilm formation upon stress. Deleting ycfR also affected outer membrane proteins and converted the cell from hydrophilic to hydrophobic, as well as increased cell aggregation fourfold. YcfR seems to be involved in the regulation of E. coli K-12 biofilm formation by decreasing cell aggregation and cell surface adhesion, by influencing the concentration of signal molecules, and by interfering with stress responses. Based on our findings, we propose that this locus be named bhsA, for influencing biofilm through hydrophobicity and stress response.     

The role of periplasmic antioxidant enzymes (superoxide dismutase and thiol peroxidase) of the Shiga toxin-producing Escherichia coli O157:H7 in the formation of biofilms

This study examined the role of the periplasmic oxidative defense proteins, copper, zinc superoxide dismutase (SodC), and thiol peroxidase (Tpx), from the Shiga toxin-producing Escherichia coli O157:H7 (STEC) in the formation of biofilms. Proteomic analyses have shown significantly higher expression levels of both periplasmic antioxidant systems (SodC and Tpx) in STEC cells grown under biofilm conditions than under planktonic conditions. An analysis of their growth phase-dependent gene expression indicated that a high level of the sodC expression occurred during the stationary phase and that the expression of the tpx gene was strongly induced only during the exponential growth phase. Exogenous hydrogen peroxide reduced the aerobic growth of the STEC sodC and tpx mutants by more than that of their parental strain. The two mutants also displayed significant reductions in their attachment to both biotic (HT-29 epithelial cell) and abiotic surfaces (polystyrene and polyvinyl chloride microplates) during static aerobic growth. However, the growth rates of both wild-type and mutants were similar under aerobic growth conditions. The formation of an STEC biofilm was only observed with the wild-type STEC cells in glass capillary tubes under continuous flow-culture conditions compared with the STEC sodC and tpx mutants. To the best of our knowledge, this is the first mutational study to show the contribution of sodC and tpx gene products to the formation of an E. coli O157:H7 biofilm. These results also suggest that these biofilms are physiologically heterogeneous and that oxidative stress defenses in both the exponential and stationary growth stages play important roles in the formation of STEC biofilms.     

Linkage between cellular adherence and biofilm formation in Escherichia coli O157:H7 EDL933

During the establishment of Escherichia coli O157:H7 infection, its capacity to adhere to host intestinal epithelial cells is the critical first step in pathogenesis. It also has the capability to form biofilms, and because both are surface activities, we sought to gain insight into a potential linkage between biofilm formation and adherence to epithelial cells. We conducted an adherence assay with 51 biofilm-negative mutants and two human epithelial cell lines, T84 and HEp2. Our results show that unlike wild-type cells, biofilm-negative mutants adhere poorly to epithelial cells. Some adhesin-negative mutants were fully competent in biofilm formation, however. Thus, biofilm-forming activity in E. coli O157:H7 EDL933 is required for adherence to T84 and HEp2 cells, but it is not sufficient.     

Role of Extracellular Structures of Escherichia coli O157:H7 in Initial Attachment to Biotic and Abiotic Surfaces

Infection by human pathogens through the consumption of fresh, minimally processed produce and solid plant-derived foods is a major concern of the U.S. and global food industries and of public health services. Enterohemorrhagic Escherichia coli O157:H7 is a frequent and potent foodborne pathogen that causes severe disease in humans. Biofilms formed by E. coli O157:H7 facilitate cross-contamination by sheltering pathogens and protecting them from cleaning and sanitation operations. The objective of this research was to determine the role that several surface structures of E. coli O157:H7 play in adherence to biotic and abiotic surfaces. A set of isogenic deletion mutants lacking major surface structures was generated. The mutant strains were inoculated onto fresh spinach and glass surfaces, and their capability to adhere was assessed by adherence assays and fluorescence microscopy methods. Our results showed that filament-deficient mutants bound to the spinach leaves and glass surfaces less strongly than the wild-type strain did. We mimicked the switch to the external environment—during which bacteria leave the host organism and adapt to lower ambient temperatures of cultivation or food processing—by decreasing the temperature from 37°C to 25°C and 4°C. We concluded that flagella and some other cell surface proteins are important factors in the process of initial attachment and in the establishment of biofilms. A better understanding of the specific roles of these structures in early stages of biofilm formation can help to prevent cross-contaminations and foodborne disease outbreaks.     

Curli fibers are required for development of biofilm architecture in Escherichia coli K-12 and enhance bacterial adherence to human uroepithelial cells

Sessile bacteria show phenotypical, biochemical, and morphological differences from their planktonic counterparts. Curli, extracellular structures important for biofilm formation, are only produced at temperatures below 30 C in Escherichia coli K-12 strains. In this report, we show that E. coli K-12 can produce curli at 37 C when grown as a biofilm community. The curli-expressing strain formed more biofilms on polyurethane sheets than the curli-deficient strain under growth temperatures of both 25 C and 37 C. Curli are required for the formation of a three-dimensional mature biofilm, with characteristic water channels and pillars of bacteria. Observations by electron microscopy revealed the presence at the surfaces of the curli-deficient mutant in biofilm of flagella and type I pili. A wild-type curli-expressing E. coli strain significantly adhered to several lines of human uroepithelial cells, more so than an isogenic curlideficient strain. The finding that curli are expressed at 37 C in biofilm and enhance bacterial adherence to mammalian host cells suggests an important role for curli in pathogenesis.     

Phosphoglycerate mutase affects Stenotrophomonas maltophilia attachment to biotic and abiotic surfaces

Stenotrophomonas maltophilia biofilm formation is of increasing medical concern, particularly for lung infections. However, the molecular mechanisms facilitating the biofilm lifestyle in S. maltophilia are poorly understood. We generated and screened a transposon mutant library for mutations that lead to altered biofilm formation compared to wild type. One of these mutations, in the gene for glycolytic enzyme phosphoglycerate mutase (gpmA), resulted in impaired attachment on abiotic and biotic surfaces. As adherence to a surface is the initial step in biofilm developmental processes, our results reveal a unique factor that could affect S. maltophilia biofilm initiation and, possibly, subsequent development.     

The role of disulfide bond isomerase A (DsbA) of Escherichia coli O157:H7 in biofilm formation and virulence

The role of periplasmic disulfide oxidoreductase DsbA in Shiga toxin-producing Escherichia coli O157:H7 (STEC) was investigated. Deletion of dsbA (DeltadsbA) significantly decreased cell motility and alkaline phosphatase activity in STEC. STEC DeltadsbA also showed greater sensitivity to menadione and under low pH conditions. Significant reductions in surface attachment to both biotic (HT-29 epithelial cells) and abiotic (polystyrene and polyvinyl chloride) surfaces were observed in STEC DeltadsbA. In addition, no biofilm formation was detected in STEC DeltadsbA compared to wild-type cells in glass capillary tubes under continuous flow-culture system conditions. In the nematode model Caenorhabditis elegans-killing assay, the deletion of dsbA in STEC resulted in attenuated virulence compared to wild-type cells. STEC DeltadsbA was also found to have a reduced ability to colonize the nematode gut. These results suggest that DsbA plays important roles in biofilm formation and virulence in STEC cells.     

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.