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Isolation and characterization of Shigella flexneri G3, capable of effective cellulosic saccharification under mesophilic conditions.

ABSTRACT: A novel Shigella strain (Shigella flexneri G3) showing high cellulolytic activity under mesophilic, anaerobic conditions was isolated and characterized. The bacterium is Gram negative, short rod shaped, and nonmotile and displays effective production of glucose, cellobiose, and other oligosaccharides from cellulose (Avicel PH-101) under optimal conditions (40°C and pH 6.5). Approximately 75% of the cellulose was hydrolyzed in modified ATCC 1191 medium containing 0.3% cellulose, and the oligosaccharide production yield and specific production rate reached 375 mg g Avicel(-1) and 6.25 mg g Avicel(-1) h(-1), respectively, after a 60-hour incubation. To our knowledge, this represents the highest oligosaccharide yield and specific rate from cellulose for mesophilic bacterial monocultures reported so far. The results demonstrate that S. flexneri G3 is capable of rapid conversion of cellulose to oligosaccharides, with potential biofuel applications under mesophilic conditions.

SUBMITTER: Wang A

PROVIDER: S-EPMC3020557 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Isolation and characterization of Shigella flexneri G3, capable of effective cellulosic saccharification under mesophilic conditions.

Wang Aijie A Gao Lingfang L Ren Nanqi N Xu Jifei J Liu Chong C Cao Guangli G Yu Hao H Liu Wenzong W Hemme Christopher L CL He Zhili Z Zhou Jizhong J

Applied and environmental microbiology 20101119 2

A novel Shigella strain (Shigella flexneri G3) showing high cellulolytic activity under mesophilic, anaerobic conditions was isolated and characterized. The bacterium is Gram negative, short rod shaped, and nonmotile and displays effective production of glucose, cellobiose, and other oligosaccharides from cellulose (Avicel PH-101) under optimal conditions (40°C and pH 6.5). Approximately 75% of the cellulose was hydrolyzed in modified ATCC 1191 medium containing 0.3% cellulose, and the oligosacc ...[more]

PMID: 21097577

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Project description:The Shigella species are Gram-negative, facultative intracellular pathogens that invade the colonic epithelium and cause significant diarrheal disease. Despite extensive research on the pathogen, a comprehensive understanding of how Shigella initiates contact with epithelial cells remains unknown. Shigella maintains many of the same Escherichia coli adherence gene operons; however, at least one critical gene component in each operon is currently annotated as a pseudogene in reference genomes. These annotations, coupled with a lack of structures upon microscopic analysis following growth in laboratory media, have led the field to hypothesize that Shigella is unable to produce fimbriae or other traditional adherence factors. Nevertheless, our previous analyses have demonstrated that a combination of bile salts and glucose induces both biofilm formation and adherence to colonic epithelial cells. The goal of this study was to perform transcriptomic and genetic analyses to demonstrate that adherence gene operons in Shigella flexneri strain 2457T are functional, despite the gene annotations. Our results demonstrate that at least three structural genes facilitate S. flexneri 2457T adherence for epithelial cell contact and biofilm formation. Furthermore, our results demonstrate that host factors, namely, glucose and bile salts at their physiological concentrations in the small intestine, offer key environmental stimuli required for adherence factor expression in S. flexneri This research may have a significant impact on Shigella vaccine development and further highlights the importance of utilizing in vivo-like conditions to study bacterial pathogenesis.IMPORTANCE Bacterial pathogens have evolved to regulate virulence gene expression at critical points in the colonization and infection processes to successfully cause disease. The Shigella species infect the epithelial cells lining the colon to result in millions of cases of diarrhea and a significant global health burden. As antibiotic resistance rates increase, understanding the mechanisms of infection is vital to ensure successful vaccine development. Despite significant gains in our understanding of Shigella infection, it remains unknown how the bacteria initiate contact with the colonic epithelium. Most pathogens harbor multiple adherence factors to facilitate this process, but Shigella was thought to have lost the ability to produce these factors. Interestingly, we have identified conditions that mimic some features of gastrointestinal transit and that enable Shigella to express adherence structural genes. This work highlights aspects of genetic regulation for Shigella adherence factors and may have a significant impact on future vaccine development.

Project description:Shigella flexneri is an intracellular human pathogen that invades colonic cells and causes bloody diarrhea. S. flexneri evolved from commensal Escherichia coli, and genome comparisons reveal that S. flexneri has lost approximately 20% of its genes through the process of pathoadaptation, including a disproportionate number of genes associated with the turnover of the nucleotide-based second messenger cyclic di-GMP (c-di-GMP); however, the remaining c-di-GMP turnover enzymes are highly conserved. c-di-GMP regulates many behavioral changes in other bacteria in response to changing environmental conditions, including biofilm formation, but this signaling system has not been examined in S. flexneri. In this study, we expressed VCA0956, a constitutively active c-di-GMP synthesizing diguanylate cyclase (DGC) from Vibrio cholerae, in S. flexneri to determine if virulence phenotypes were regulated by c-di-GMP. We found that expressing VCA0956 in S. flexneri increased c-di-GMP levels, and this corresponds with increased biofilm formation and reduced acid resistance, host cell invasion, and plaque size. We examined the impact of VCA0956 expression on the S. flexneri transcriptome and found that genes related to acid resistance were repressed, and this corresponded with decreased survival to acid shock. We also found that individual S. flexneri DGC mutants exhibit reduced biofilm formation and reduced host cell invasion and plaque size, as well as increased resistance to acid shock. This study highlights the importance of c-di-GMP signaling in regulating S. flexneri virulence phenotypes. IMPORTANCE The intracellular human pathogen Shigella causes dysentery, resulting in as many as one million deaths per year. Currently, there is no approved vaccine for the prevention of shigellosis, and the incidence of antimicrobial resistance among Shigella species is on the rise. Here, we explored how the widely conserved c-di-GMP bacterial signaling system alters Shigella behaviors associated with pathogenesis. We found that expressing or removing enzymes associated with c-di-GMP synthesis results in changes in Shigella's ability to form biofilms, invade host cells, form lesions in host cell monolayers, and resist acid stress.

Project description:BackgroundShigella flexneri is the major cause of shigellosis in the developing countries. The O-antigen component of the lipopolysaccharide is one of the key virulence determinants required for the pathogenesis of S. flexneri. The glucosyltransferase and/or acetyltransferase genes responsible for the modification of the O-antigen are encoded by temperate serotype converting bacteriophage present in the S. flexneri genome. Several serotype converting phages have previously been isolated and characterized, however, attempts to isolate a serotype converting phage which encodes the modification genes of serotypes 4a strain have not been successful.ResultsIn this study, a novel temperate serotype converting bacteriophage SfIV was isolated. Lysogenisation of phage SfIV converted serotype Y strain to serotype 4a. Electron microscopy indicated that SfIV belongs to Myoviridae family. The 39,758 bp genome of phage SfIV encompasses 54 open reading frames (orfs). Protein level comparison of SfIV with other serotype converting phages of S. flexneri revealed that SfIV is similar to phage SfII and SfV. The comparative analysis also revealed that SfIV phage contained five proteins which were not found in any other phages of S. flexneri. These proteins were: a tail fiber assembly protein, two hypothetical proteins with no clear function, and two other unknown proteins which were encoded by orfs present on a moron, that presumably got introduced in SfIV genome from another species via a transposon. These unique proteins of SfIV may play a role in the pathogenesis of the host.ConclusionsThis study reports the isolation and complete genome sequence analysis of bacteriophage SfIV. The SfIV phage has a host range significantly different from the other phages of Shigella. Comparative genome analysis identified several proteins unique to SfIV, which may potentially be involved in the survival and pathogenesis of its host. These findings will further our understanding on the evolution of these phages, and will also facilitate studies on development of new phage vectors and therapeutic agents to control infections caused by S. flexneri.

Dataset Information

Isolation and characterization of Shigella flexneri G3, capable of effective cellulosic saccharification under mesophilic conditions.

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Isolation and characterization of Shigella flexneri G3, capable of effective cellulosic saccharification under mesophilic conditions.

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