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Archives of Microbiology (v.194, #6)

Inhibition and dispersal of Agrobacterium tumefaciens biofilms by a small diffusible Pseudomonas aeruginosa exoproduct(s) by Michael E. Hibbing; Clay Fuqua (pp. 391-403).

Environmental biofilms often contain mixed populations of different species. In these dense communities, competition between biofilm residents for limited nutrients such as iron can be fierce, leading to the evolution of competitive factors that affect the ability of competitors to grow or form biofilms. We have discovered a compound(s) present in the conditioned culture fluids of Pseudomonas aeruginosa that disperses and inhibits the formation of biofilms produced by the facultative plant pathogen Agrobacterium tumefaciens. The inhibitory activity is strongly induced when P. aeruginosa is cultivated in iron-limited conditions, but it does not function through iron sequestration. In addition, the production of the biofilm inhibitory activity is not regulated by the global iron regulatory protein Fur, the iron-responsive extracytoplasmic function σ factor PvdS, or three of the recognized P. aeruginosa quorum-sensing systems. In addition, the compound(s) responsible for the inhibition and dispersal of A. tumefaciens biofilm formation is likely distinct from the recently identified P. aeruginosa dispersal factor, cis-2-decenoic acid (CDA), as dialysis of the culture fluids showed that the inhibitory compound was larger than CDA and culture fluids that dispersed and inhibited biofilm formation by A. tumefaciens had no effect on biofilm formation by P. aeruginosa.

Keywords: Biofilms; Inhibition; Dispersal; Iron

Deletion of glucose-inhibited division (gidA) gene alters the morphological and replication characteristics of Salmonella enterica Serovar typhimurium by Daniel C. Shippy; Joseph A. Heintz; Ralph M. Albrecht; Nicholas M. Eakley; Amin A. Fadl (pp. 405-412).

Salmonella is an important food-borne pathogen that continues to plague the United States food industry. Characterization of bacterial factors involved in food-borne illnesses could help develop new ways to control salmonellosis. We have previously shown that deletion of glucose-inhibited division gene (gidA) significantly altered the virulence potential of Salmonella in both in vitro and in vivo models of infection. Most importantly, the gidA mutant cells displayed a filamentous morphology compared to the wild-type Salmonella cells. In our current study, we investigated the role of GidA in Salmonella cell division using fluorescence and electron microscopy, transcriptional, and proteomic assays. Scanning electron microscopy data indicated a filamentous morphology with few constrictions in the gidA mutant cells. The filamentation of the gidA mutant cells is most likely due to the defect in chromosome segregation, with little to no sign of septa formation observed using fluorescence and transmission electron microscopy. Furthermore, deletion of gidA altered the expression of many genes and proteins responsible for cell division and chromosome segregation as indicated by global transcriptional profiling and semi-quantitative western blot analysis. Taken together, our data indicate GidA as a potential regulator of Salmonella cell division genes.

Keywords: Salmonella ; GidA; Cell division; Electron microscopy

Erratum to: Deletion of glucose-inhibited division (gidA) gene alters the morphological and replication characteristics of Salmonella enterica Serovar typhimurium by Daniel C. Shippy; Joseph A. Heintz; Ralph M. Albrecht; Nicholas M. Eakley; Ashok K. Chopra; Amin A. Fadl (pp. 413-413).

The biosynthesis of the polyether antibiotic nanchangmycin is controlled by two pathway-specific transcriptional activators by Qing Yu; Aiqin Du; Tiangang Liu; Zixin Deng; Xinyi He (pp. 415-426).

The nanchangmycin (NAN) produced by Streptomyces nanchangensis NS3226 is a polyether antibiotic resembling monensin in their gene clusters and the chemical structures. They can inhibit gram-positive bacteria and be a growth promoter for ruminants. Within the nanchangmycin gene cluster (nan), we identified that two SARP-family regulatory genes, nanR1 and nanR2, were both required to activate the transcription of all nan polyketide genes. Overexpression of NanR1 and NanR2 in wild-type increase NAN yields by at least three folds. Bioinformatic analysis of the immediate upstream DNA sequence of each nan gene and quantitative real-time RT-PCR analysis of the nan operons identified five putative SARP binding sites. Moreover, deletion of an AraC-family repressor gene nanR4 increased expression of NanR1 and R2 and led to a threefold increase in NAN production.

Keywords: SARP; Nanchangmycin; Streptomyces; Regulation

The anisin1 gene encodes a defensin-like protein and supports the fitness of Aspergillus nidulans by Andrea Eigentler; István Pócsi; Florentine Marx (pp. 427-437).

In the genome of Aspergillus nidulans, a defensin-like protein, Anisin1, was annotated with high homology to the mosquito defensin AaDefA1. So far, no studies exist on defensins from filamentous ascomycetes. Therefore, we characterized the anisin1 gene in A. nidulans and generated a deletion mutant, which suffered from a defect in mitospore development and produced less conidia at 42°C compared to the reference strain. In surface cultures of A. nidulans wild type, the anisin1 expression correlated with that of the central regulator for asexual development, brlA, and with the major scavanger of H₂O₂ stress, catB, which is indicative for cell differentiation in developing fungi. Interestingly, brlA and anisin1 expressions were deregulated in a ΔsrrA strain that covers a central role in the histidine-to-aspartate (His-Asp) phosphorelay signaling pathway and shows impaired asexual development and H₂O₂ detoxification. In submers cultures of A. nidulans wild type and other mutants of the His-Asp phosphorelay signaling pathway, anisin1 was repressed, but derepressed in a ΔsrrA background, and anisin1 transcription was further increased in this mutant by H₂O₂ addition. We therefore conclude that the secreted protein Anisin1 contributes to the optimal development of A. nidulans and we further propose that it has a sensing/signaling function for elevated H₂O₂ levels.

Keywords: Defensin; Asexual development; Oxidative stress; Aspergillus nidulans

Characterization of a new quorum sensing regulator luxT and its roles in the extracellular protease production, motility, and virulence in fish pathogen Vibrio alginolyticus by Huan Liu; Dan Gu; Xiaodan Cao; Qin Liu; Qiyao Wang; Yuanxing Zhang (pp. 439-452).

Vibrio alginolyticus, an opportunistic pathogen that causes vibriosis in miscellaneous fish species, has brought about serious economic damage to the mariculture industry in South China. The mechanism of virulence regulation in V. alginolyticus is yet not known except a Vibrio harveyi-like quorum sensing (QS) system that is established to manipulate the expression of diverse genes including those encoding virulence determinants. In this study, a new TetR family QS regulator, luxT, was identified and characterized in V. alginolyticus. The transcription of luxT gene was cell density dependent and was positively regulated by LuxU, an established QS component relaying the signal from three paralleled QS regulatory systems in V. harveyi. In addition, luxT positively regulated both luxO at transcriptional level and luxR at post-transcriptional level, which is thoroughly different from the established QS regulation mode in V. harveyi and Vibrio vulnificus. The mutant of luxT deletion produced markedly decreased total extracellular proteases and reduced motility ability compared to the wild type and the complemented strain luxT ⁺. The fish infection results indicated that mutation of luxT led to marginal attenuation in the virulence of V. alginolyticus, suggesting that LuxT might play a role in the fine-tuning of the virulence via QS in V. alginolyticus.

Keywords: Extracellular protease; luxT ; Motility; Quorum sensing; V. alginolyticus

Characterization of the BaeSR two-component system from Salmonella Typhimurium and its role in ciprofloxacin-induced mdtA expression by P. Guerrero; B. Collao; E. H. Morales; I. L. Calderón; F. Ipinza; S. Parra; C. P. Saavedra; F. Gil (pp. 453-460).

Two-component systems are one of the most prevalent mechanisms by which bacteria sense, respond and adapt to changes in their environment. The activation of a sensor histidine kinase leads to autophosphorylation of a conserved histidine residue followed by transfer of the phosphoryl group to a cognate response regulator in an aspartate residue. The search for antibiotics that inhibit molecular targets has led to study prokaryotic two-component systems. In this study, we characterized in vitro and in vivo the BaeSR two-component system from Salmonella Typhimurium and evaluated its role in mdtA regulation in response to ciprofloxacin treatment. We demonstrated in vitro that residue histidine 250 is essential for BaeS autophosphorylation and aspartic acid 61 for BaeR transphosphorylation. By real-time PCR, we showed that mdtA activation in the presence of ciprofloxacin depends on both members of this system and that histidine 250 of BaeS and aspartic acid 61 of BaeR are needed for this. Moreover, the mdtA expression is directly regulated by binding of BaeR at the promoter region, and this interaction is enhanced when the protein is phosphorylated. In agreement, a BaeR mutant unable to phosphorylate at aspartic acid 61 presents a lower affinity with the mdtA promoter.

Keywords: Two-component system; BaeSR; Ciprofloxacin

Electron transport and oxidative stress in Zymomonas mobilis respiratory mutants by Inese Strazdina; Zane Kravale; Nina Galinina; Reinis Rutkis; Robert K. Poole; Uldis Kalnenieks (pp. 461-471).

The ethanol-producing bacterium Zymomonas mobilis is of great interest from a bioenergetic perspective because, although it has a very high respiratory capacity, the respiratory system does not appear to be primarily required for energy conservation. To investigate the regulation of respiratory genes and function of electron transport branches in Z. mobilis, several mutants of the common wild-type strain Zm6 (ATCC 29191) were constructed and analyzed. Mutant strains with a chloramphenicol-resistance determinant inserted in the genes encoding the cytochrome b subunit of the bc ₁ complex (Zm6-cytB), subunit II of the cytochrome bd terminal oxidase (Zm6-cydB), and in the catalase gene (Zm6-kat) were constructed. The cytB and cydB mutants had low respiration capacity when cultivated anaerobically. Zm6-cydB lacked the cytochrome d absorbance at 630 nm, while Zm6-cytB had very low spectral signals of all cytochromes and low catalase activity. However, under aerobic growth conditions, the respiration capacity of the mutant cells was comparable to that of the parent strain. The catalase mutation did not affect aerobic growth, but rendered cells sensitive to hydrogen peroxide. Cytochrome c peroxidase activity could not be detected. An upregulation of several thiol-dependent oxidative stress-protective systems was observed in an aerobically growing ndh mutant deficient in type II NADH dehydrogenase (Zm6-ndh). It is concluded that the electron transport chain in Z. mobilis contains at least two electron pathways to oxygen and that one of its functions might be to prevent endogenous oxidative stress.

Keywords: Zymomonas mobilis ; Respiratory chain; Cytochrome bd ; Cytochrome bc ₁

Transcriptome analysis of pellicle formation of Shewanella oneidensis by Yili Liang; Haichun Gao; Xue Guo; Jingrong Chen; Guanzhou Qiu; Zhili He; Jizhong Zhou; Xueduan Liu (pp. 473-482).

Although the pellicle is one of the major growth modes of microorganisms, the metabolic features of pellicle cells and the determinative factors for pellicle formation are largely unknown. In recent years, biofilm development of Shewanella oneidensis, an important model organism for bioremediation studies, has been extensively studied. In this paper, a transcriptional profiling of pellicle cells relative to planktonic cells indicated that cells in pellicles were more metabolically active than the planktonic cells. Most notably, up-transcription of general secretion system proteins and iron/heme uptake and transport proteins was observed in pellicle cells. Unexpectedly, neither the hmuT nor hugA heme transport mutant exhibited a significant defect in pellicle formation. Expectedly, three type I secretion system mutants were severely deficient in pellicle formation, suggesting an essential role of these proteins.

Keywords: Pellicle; Shewanella oneidensis ; Type I secretion system

Effect of salinity on nitrogenase activity and composition of the active diazotrophic community in intertidal microbial mats by Ina Severin; Veronique Confurius-Guns; Lucas J. Stal (pp. 483-491).

Microbial mats are often found in intertidal areas experiencing a large range of salinities. This study investigated the effect of changing salinities on nitrogenase activity and on the composition of the active diazotrophic community (nifH transcript libraries) of three types of microbial mats situated along a littoral gradient. All three mat types exhibited highest nitrogenase activity at salinities close to ambient seawater or lower. The response to lower or higher salinity was strongest in mats higher up in the littoral zone. Changes in nitrogenase activity as the result of exposure to different salinities were accompanied by changes in the active diazotrophic community. The two stations higher up in the littoral zone showed nifH expression by Cyanobacteria (Oscillatoriales and Chroococcales) and Proteobacteria (Gammaproteobacteria and Deltaproteobacteria). At these stations, a decrease in the relative contribution of Cyanobacteria to the nifH transcript libraries was observed at increasing salinity coinciding with a decrease in nitrogenase activity. The station at the low water mark showed low cyanobacterial contribution to nifH transcript libraries at all salinities but an increase in deltaproteobacterial nifH transcripts under hypersaline conditions. In conclusion, increased salinities caused decreased nitrogenase activity and were accompanied by a lower proportion of cyanobacterial nifH transcripts.

Keywords: Salinity; Microbial mat; Nitrogen fixation; Cyanobacteria ; Proteobacteria

Characterization of Edwardsiella tarda rpoN: roles in σ⁷⁰ family regulation, growth, stress adaption and virulence toward fish by Keping Wang; Enfu Liu; Shanshan Song; Xiaobo Wang; Yunxia Zhu; Jiang Ye; Huizhan Zhang (pp. 493-504).

Edwardsiella tarda EIB202, a Gram-negative pathogen with strong virulence, is an opportunistic pathogen capable of causing edwardsiellosis with high mortality to fish. Alternative sigma factor 54 (RpoN) is an important regulator of virulence and stress resistance genes in many bacterial species and mainly responsible for transcription of genes in nitrogen utilization. In this study, the in-frame rpoN deletion mutant was constructed to analyze the function of RpoN in Edwardsiella tarda firstly. Compared to the wild-type and complemented strain rpoN ⁺, the ΔrpoN was impaired in terms of the ability to survive under oxidative stress, osmotic stress and acid resistance, as well as the growth in Luria–Bertani medium, demonstrating essential roles of RpoN in stress resistance and nitrogen utilization. In addition, the ΔrpoN displayed markedly decreased biofilm formation and chondroitinase activity and was attenuated in virulence reflected in the increased median lethal dose value and extended infection cycle. Real-time polymerase chain reaction revealed that the expression levels of σ⁷⁰ class changed in varying degrees in the rpoN mutant. Especially, the expression levels of rpoS and fliA were down-regulated 4.1-fold and 7.9-fold in stationary phase in comparison with the wild type, respectively. Furthermore, two differential expression genes, znuA and flhC, were detected in the wild type and ΔrpoN using the method of differential display reverse transcription PCR.

Keywords: Edwardsiella tarda ; rpoN ; Biofilm formation; Virulence

Biochemical characterisation of the chlamydial MurF ligase, and possible sequence of the chlamydial peptidoglycan pentapeptide stem by Delphine Patin; Julieanne Bostock; Ian Chopra; Dominique Mengin-Lecreulx; Didier Blanot (pp. 505-512).

Chlamydiaceae are obligate intracellular bacteria that do not synthesise detectable peptidoglycan although they possess an almost complete arsenal of genes encoding peptidoglycan biosynthetic activities. In this paper, the murF gene from Chlamydia trachomatis was shown to be capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-tripeptide:d-Ala-d-Ala ligase activity. Recombinant MurF from C. trachomatis was overproduced and purified from E. coli. It exhibited ATP-dependent UDP-MurNAc-X-γ-d-Glu-meso-A₂pm:d-Ala-d-Ala ligase activity in vitro. No significant difference of kinetic parameters was seen when X was l-Ala, l-Ser or Gly. The l-Lys-containing UDP-MurNAc-tripeptide was a poorer substrate as compared to the meso-A₂pm-containing one. Based on the respective substrate specificities of the chlamydial MurC, MurE, MurF and Ddl enzymes, a sequence l-Ala/l-Ser/Gly-γ-d-Glu-meso-A₂pm-d-Ala-d-Ala is expected for the chlamydial pentapeptide stem, with Gly at position 1 being less likely.

Keywords: Chlamydiaceae ; C. trachomatis ; MurF; Mur ligases; Peptidoglycan

Microbial biodiversity in a Malaysian oil field and a systematic comparison with oil reservoirs worldwide by Dongmei Li; David J. Midgley; Jason P. Ross; Yalchin Oytam; Guy C. J. Abell; Herbert Volk; Wan Ata Wan Daud; Philip Hendry (pp. 513-523).

Microbial diversity within formation water and oil from two compartments in Bokor oil reservoir from a Malaysian petroleum oil field was examined. A total of 1,056 16S rRNA gene clones were screened from each location by amplified ribosomal DNA restriction analysis. All samples were dominated by clones affiliated with Marinobacter, some novel Deferribacteraceae genera and various clones allied to the Methanococci. In addition, either Marinobacterium- or Pseudomonas-like operational taxonomic units were detected from either compartment. A systematic comparison with the existing pertinent studies was undertaken by analysing the microbial amplicons detected and the PCR primers used. The analyses demonstrated that bacterial communities were site specific, while Archaea co-occurred more frequently. Amplicons related to Marinobacter, Marinobacterium and Pseudomonas were detected in a number of the studies examined, suggesting they may be ubiquitous members in oil reservoirs. Further analysis of primers used in those studies suggested that most primer pairs had fairly broad but low matches across the bacterial and archaeal domains, while a minority had selective matches to certain taxa or low matches to all the microbial taxa tested. Thus, it indicated that primers may play an important role in determining which taxa would be detected.

Keywords: Petroleum; Formation water; Oil; Biodiversity; Primer

Analysis of the cell surface layer ultrastructure of the oral pathogen Tannerella forsythia by Gerhard Sekot; Gerald Posch; Yoo Jin Oh; Sonja Zayni; Harald F. Mayer; Dietmar Pum; Paul Messner; Peter Hinterdorfer; Christina Schäffer (pp. 525-539).

The Gram-negative oral pathogen Tannerella forsythia is decorated with a 2D crystalline surface (S-) layer, with two different S-layer glycoprotein species being present. Prompted by the predicted virulence potential of the S-layer, this study focused on the analysis of the arrangement of the individual S-layer glycoproteins by a combination of microscopic, genetic, and biochemical analyses. The two S-layer genes are transcribed into mRNA and expressed into protein in equal amounts. The S-layer was investigated on intact bacterial cells by transmission electron microscopy, by immune fluorescence microscopy, and by atomic force microscopy. The analyses of wild-type cells revealed a distinct square S-layer lattice with an overall lattice constant of 10.1 ± 0.7 nm. In contrast, a blurred lattice with a lattice constant of 9.0 nm was found on S-layer single-mutant cells. This together with in vitro self-assembly studies using purified (glyco)protein species indicated their increased structural flexibility after self-assembly and/or impaired self-assembly capability. In conjunction with TEM analyses of thin-sectioned cells, this study demonstrates the unusual case that two S-layer glycoproteins are co-assembled into a single S-layer. Additionally, flagella and pilus-like structures were observed on T. forsythia cells, which might impact the pathogenicity of this bacterium.

Keywords: Tannerella forsythia ; S-layer; Ultrastructure; Glycoprotein; Transmission electron microscopy; Atomic force microscopy

Role of anionic charges of osmoregulated periplasmic glucans of Salmonella enterica serovar Typhimurium SL1344 in mice virulence by Arvind A. Bhagwat; Porteen Kannan; Yi Ning Leow; Mahesh Dharne; Allen Smith (pp. 541-548).

opgB gene of Salmonella enterica serovar Typhimurium was identified earlier in a genome-wide screen for mice virulence (Valentine et al. in Infect Immun 66:3378–3383, 1998). Although mutation in opgB resulted in avirulent Salmonella strain, how this gene contributes to pathogenesis remains unclear. Based on DNA homology, opgB is predicted to be responsible for adding phosphoglycerate residues to osmoregulated periplasmic glucans (OPGs) giving them anionic characteristics. In Escherichia coli, yet another gene, opgC, is also reported to contribute to anionic characteristics of OPGs by adding succinic acid residues. We constructed opgB, opgC, and opgBC double mutants of S. enterica serovar Typhimurium strain SL1344. As predicted opgBC mutant synthesized neutral OPGs that were devoid of any anionic substituents. However, opgB, opgC, and opgBC mutations had no significant impact on mice virulence as well as on competitive organ colonization. In low osmotic conditions, opgB, opgC, and opgBC mutants exhibited delay in growth initiation in the presence of sodium deoxycholate. Anionic substituents of OPGs from Salmonella although appear to be needed to overcome resistance of deoxycholate in hypoosmotic growth media, no evidence was found for their role in mice virulence.

Keywords: Periplasmic glucans ; Low osmolarity; Bile salt sensitivity; Salmonellosis; Food microbiology; Microbial food safety

Comparative analysis of a cryptic thienamycin-like gene cluster identified in Streptomyces flavogriseus by genome mining by Gloria Blanco (pp. 549-555).

In silico database searches allowed the identification in the S. flavogriseus ATCC 33331 genome of a carbapenem gene cluster highly related to the S. cattleya thienamycin one. This is the second cluster found for a complex highly substituted carbapenem. Comparative analysis revealed that both gene clusters display a high degree of synteny in gene organization and in protein conservation. Although the cluster appears to be silent under our laboratory conditions, the putative metabolic product was predicted from bioinformatics analyses using sequence comparison tools. These data, together with previous reports concerning epithienamycins production by S. flavogriseus strains, suggest that the cluster metabolic product might be a thienamycin-like carbapenem, possibly the epimeric epithienamycin. This finding might help in understanding the biosynthetic pathway to thienamycin and other highly substituted carbapenems. It also provides another example of genome mining in Streptomyces sequenced genomes as a powerful approach for novel antibiotic discovery.

Keywords: Carbapenem; β-Lactam; Epithienamycin; Antibiotic; Streptomyces cattleya

4-Hydroxyphenylglycine biosynthesis in Herpetosiphon aurantiacus: a case of gene duplication and catalytic divergence by Stephan Kastner; Sebastian Müller; Lavanya Natesan; Gabriele M. König; Reinhard Guthke; Markus Nett (pp. 557-566).

The nonproteinogenic amino acid 4-hydroxyphenylglycine (HPG) arises from the diversion of the tyrosine degradation pathway into secondary metabolism, and its biosynthesis requires a set of three enzymes. The gene cassette for HPG biosynthesis is widely spread in actinomycete bacteria, which incorporate the amino acid as a building block into various peptide antibiotics, but it has never been reported from another taxonomic group of eubacteria. A genome mining study has now revealed a putative HPG pathway in the predatory bacterium Herpetosiphon aurantiacus, which is phylogenetically distinct from Actinomycetes. Anomalies in the active center of one annotated key enzyme raised questions about the true product of this pathway, prompting an in vitro reconstitution attempt. This study confirmed the capability of H. aurantiacus for HPG production. Sequence analysis of the aberrant 4-hydroxymandelate synthase refines the existing model on the catalytic differentiation of iron(II)-dependent dioxygenases. Furthermore, we report a comprehensive analysis on the phylogeny of these enzymes, which sheds light on the evolution of paralogous gene sets and the ensuing metabolic diversity in a barely studied bacterium.

Keywords: Herpetosiphon ; Biosynthesis; Dioxygenase; 4-Hydroxyphenylglycine; Phylogeny; Gene duplication

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Archives of Microbiology (v.194, #6)