Timothy R. Hoover

Novel features of the polysaccharide-digesting gliding bacterium Flavobacterium johnsoniae as revealed by genome sequence analysis

ABSTRACT The 6.10-Mb genome sequence of the aerobic chitin-digesting gliding bacterium Flavobacterium johnsoniae (phylum Bacteroidetes) is presented. F. johnsoniae is a model organism for studies of bacteroidete gliding motility, gene regulation, and biochemistry. The mechanism of F. johnsoniae gliding is novel, and genome analysis confirms that it does not involve well-studied motility organelles, such as flagella or type IV pili. The motility machinery is composed of Gld proteins in the cell envelope that are thought to comprise the “motor” and SprB, which is thought to function as a cell surface adhesin that is propelled by the motor. Analysis of the genome identified genes related to sprB that may encode alternative adhesins used for movement over different surfaces. Comparative genome analysis revealed that some of the gld and spr genes are found in nongliding bacteroidetes and may encode components of a novel protein secretion system. F. johnsoniae digests proteins, and 125 predicted peptidases were identified. F. johnsoniae also digests numerous polysaccharides, and 138 glycoside hydrolases, 9 polysaccharide lyases, and 17 carbohydrate esterases were predicted. The unexpected ability of F. johnsoniae to digest hemicelluloses, such as xylans, mannans, and xyloglucans, was predicted based on the genome analysis and confirmed experimentally. Numerous predicted cell surface proteins related to Bacteroides thetaiotaomicron SusC and SusD, which are likely involved in binding of oligosaccharides and transport across the outer membrane, were also identified. Genes required for synthesis of the novel outer membrane flexirubin pigments were identified by a combination of genome analysis and genetic experiments. Genes predicted to encode components of a multienzyme nonribosomal peptide synthetase were identified, as were novel aspects of gene regulation. The availability of techniques for genetic manipulation allows rapid exploration of the features identified for the polysaccharide-digesting gliding bacteroidete F. johnsoniae.

Transcriptional regulation at a distance in bacteria

Transcriptional enhancers are cis-acting DNA elements that are binding sites for regulatory proteins and function at large distances from promoter elements to stimulate transcription. Once thought to be unique to eukaryotes, enhancer-like elements have been discovered in a wide variety of bacteria. The regulatory proteins that bind to these bacterial enhancers must contact RNA polymerase to activate transcription. In principle, interactions between bacterial enhancer-binding proteins and RNA polymerase can occur by either DNA looping or tracking of the enhancer-binding protein along the DNA. Paradigms for each of these methods are found in bacterial systems. Activators of sigma(54)-RNA polymerase holoenzyme contact polymerase by DNA looping, while bacteriophage T4 gp45 functions as a sliding clamp that tracks along DNA until it engages RNA polymerase. Significant advances have been made over the last few years towards understanding the mechanisms by which bacterial enhancer-binding proteins activate transcription, but important aspects of these mechanisms are still poorly defined.

Insights into the complex regulation of rpoS in Borrelia burgdorferi

Co‐ordinated regulation of gene expression is required for the transmission and survival of Borrelia burgdorferi in different hosts. The sigma factor RpoS (σS), as regulated by RpoN (σ54), has been shown to regulate key virulence factors (e.g. OspC) required for these processes. As important, multiple signals (e.g. temperature, pH, cell density, oxygen) have been shown to increase the expression of σS‐dependent genes; however, little is known about the signal transduction mechanisms that modulate the expression of rpoS. In this report we show that: (i) rpoS has a σ54‐dependent promoter that requires Rrp2 to activate transcription; (ii) Rrp2Δ123, a constitutively active form of Rrp2, activated σ54‐dependent transcription of rpoS/P‐lacZ reporter constructs in Escherichia coli; (iii) quantitative reverse transcription polymerase chain reaction (QRT‐PCR) experiments with reporter cat constructs in B. burgdorferi indicated that Rrp2 activated transcription of rpoS in an enhancer‐independent fashion; and finally, (iv) rpoN is required for cell density‐ and temperature‐dependent expression of rpoS in B. burgdorferi, but histidine kinase Hk2, encoded by the gene immediately upstream of rrp2, is not essential. Based on these findings, a model for regulation of rpoS has been proposed which provides mechanisms for multiple signalling pathways to modulate the expression of the σS regulon in B. burgdorferi.

Role of integration host factor in stimulating transcription from the σ54-dependent nifH promoter☆

In a wide variety of nitrogen-fixing organisms among the Purple Bacteria (large division of Gram-negative bacteria) the nitrogen fixation (nif) operons are transcribed by an alternative holoenzyme form of RNA polymerase, sigma 54-holoenzyme. Transcription depends on the activator protein NIFA (nitrogen fixation protein A), which catalyzes isomerization of closed complexes between this polymerase and a promoter to transcriptionally productive open complexes. NIFA-mediated activation of transcription from the nifH promoter of Klebsiella pneumoniae is greatly stimulated by the integration host factor IHF, which binds to a site between the upstream binding site for NIFA and the promoter, and bends the DNA. IHF fails to stimulate activation of transcription from this promoter by another activator of sigma 54-holoenzyme, NTRC (nitrogen regulatory protein C), which lacks a specific binding site in the nifH promoter region. As predicted, if the IHF-induced bend facilitates interaction between NIFA and sigma 54-holoenzyme, substitution of an NTRC-binding site for the NIFA-binding site allowed IHF to stimulate NTRC-mediated activation of transcription from the nifH promoter. The stimulation was of the same order of magnitude as that for NIFA in the native configuration of the promoter-regulatory region (up to 20-fold). With purified NTRC and the substitution construct we could demonstrate that stimulation by IHF in a purified transcription system was comparable to that in a crude coupled transcription-translation system, indicating that the stimulation in the crude system could be accounted for by IHF. The IHF stimulation was observed on linear as well as supercoiled templates, indicating that the geometric requirements are relatively simple. We have attempted to visualize the arrangement of proteins on DNA fragments carrying the nifH promoter-regulatory region of K. pneumoniae by electron microscopy. IHF stimulated NIFA-mediated activation of transcription from the nifH and nifD promoters of Bradyrhizobium japonicum and less so from the nifH promoters of Rhizobium meliloti and Thiobacillus ferrooxidans, consistent with previous observations that stimulation is greatest at promoters that are weak binding sites for sigma 54-holoenzyme in closed complexes.

Synergistic transcriptional activation by one regulatory protein in response to two metabolites

BenM is a LysR-type bacterial transcriptional regulator that controls aromatic compound degradation in Acinetobacter sp. ADP1. Here, in vitro transcription assays demonstrated that two metabolites of aromatic compound catabolism, benzoate and cis,cis-muconate, act synergistically to activate gene expression. The level of BenM-regulated benA transcription was significantly higher in response to both compounds than the combined levels due to each alone. These compounds also were more effective together than they were individually in altering the DNase I footprint patterns of BenM-DNA complexes. This type of dual-inducer synergy provides great potential for rapid and large modulations of gene expression and may represent an important, and possibly widespread, feature of transcriptional control.

Deciphering bacterial flagellar gene regulatory networks in the genomic era.

Synthesis of the bacterial flagellum is a complex process involving dozens of structural and regulatory genes. Assembly of the flagellum is a highly-ordered process, and in most flagellated bacteria the structural genes are expressed in a transcriptional hierarchy that results in the products of these genes being made as they are needed for assembly. Temporal regulation of the flagellar genes is achieved through sophisticated regulatory networks that utilize checkpoints in the flagellar assembly pathway to coordinate expression of flagellar genes. Traditionally, flagellar transcriptional hierarchies are divided into various classes. Class I genes, which are the first genes expressed, encode a master regulator that initiates the transcriptional hierarchy. The master regulator activates transcription a set of structural and regulatory genes referred to as class II genes, which in turn affect expression of subsequent classes of flagellar genes. We review here the literature on the expression and activity of several known master regulators, including FlhDC, CtrA, VisNR, FleQ, FlrA, FlaK, LafK, SwrA, and MogR. We also examine the Department of Energy Joint Genomes Institute database to make predictions about the distribution of these regulators. Many bacteria employ the alternative sigma factors sigma(54) and/or sigma(28) to regulate transcription of later classes of flagellar genes. Transcription by sigma(54)-RNA polymerase holoenzyme requires an activator, and we review the literature on the sigma(54)-dependent activators that control flagellar gene expression in several bacterial systems, as well as make predictions about other systems that may utilize sigma(54) for flagellar gene regulation. Finally, we review the prominent systems that utilize sigma(28) and its antagonist, the anti-sigma(28) factor FlgM, along with some systems that utilize alternative mechanisms for regulating flagellar gene expression.

A conserved region in the σ54-dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation

Rhizobium melioti DctD activates transcription from the dctA promoter by catalysing the isomerization of closed complexes between σ54‐RNA polymerase holoenzyme and the promoter to open complexes. DctD must make productive contact with σ54‐holoenzyme and hydrolyse ATP to catalyse this isomerization. To define further the activation process, we sought to isolate mutants of DctD that had reduced affinities for σ54‐holoenzyme. Mutagenesis was confined to the well‐conserved C3 region of the protein, which is required for coupling ATP hydrolysis to open complex formation in σ54‐dependent activators. Mutant forms of DctD that failed to activate transcription and had substitutions in the C‐terminal half of the C3 region were efficiently cross‐linked to σ54 and the β‐subunit of RNA polymerase, suggesting that they bound normally to σ54‐holoenzyme. In contrast, some mutant forms of DctD with amino acid substitutions in the N‐terminal half of the C3 region had reduced affinities for σ54 and the β‐subunit in the cross‐linking assay. These data suggest that the N‐terminal half of the C3 region of DctD contains a site that may contact σ54‐holoenzyme during open complex formation.

Rhizobium meliloti DctD, a σ54‐dependent transcriptional activator, may be negatively controlled by a subdomain in the C‐terminal end of its two‐component receiver module

Rhizobium meliloti DctD is believed to have three functional domains: an N‐terminal, two‐component receiver domain; and like other σ54‐dependent activators, C‐terminal and central domains for DNA binding and transcription activation. We have characterized a progressive series of M‐terminal deletions of R meliloti DctD. The N‐terminal domain was not needed for binding the dctA upstream activation sequence. Only 25% of the C‐terminal end of the receiver domain was needed to significantly inhibit the central domain, and proteins lacking up to 60% of the N‐terminal end of the receiver domain were‘inducible’in R. meliloti cells. We hypothesize that the W‐terminal two‐thirds of the DctD receiver domain augments and controls an adjacent subdomain for inhibiting the central domain.

Comparative Genomic Evidence for a Close Relationship between the Dimorphic Prosthecate Bacteria Hyphomonas neptunium and Caulobacter crescentus

The dimorphic prosthecate bacteria (DPB) are alpha-proteobacteria that reproduce in an asymmetric manner rather than by binary fission and are of interest as simple models of development. Prior to this work, the only member of this group for which genome sequence was available was the model freshwater organism Caulobacter crescentus. Here we describe the genome sequence of Hyphomonas neptunium, a marine member of the DPB that differs from C. crescentus in that H. neptunium uses its stalk as a reproductive structure. Genome analysis indicates that this organism shares more genes with C. crescentus than it does with Silicibacter pomeroyi (a closer relative according to 16S rRNA phylogeny), that it relies upon a heterotrophic strategy utilizing a wide range of substrates, that its cell cycle is likely to be regulated in a similar manner to that of C. crescentus, and that the outer membrane complements of H. neptunium and C. crescentus are remarkably similar. H. neptunium swarmer cells are highly motile via a single polar flagellum. With the exception of cheY and cheR, genes required for chemotaxis were absent in the H. neptunium genome. Consistent with this observation, H. neptunium swarmer cells did not respond to any chemotactic stimuli that were tested, which suggests that H. neptunium motility is a random dispersal mechanism for swarmer cells rather than a stimulus-controlled navigation system for locating specific environments. In addition to providing insights into bacterial development, the H. neptunium genome will provide an important resource for the study of other interesting biological processes including chromosome segregation, polar growth, and cell aging.

Survival in nuclear waste, extreme resistance, and potential applications gleaned from the genome sequence of Kineococcus radiotolerans SRS30216.

Kineococcus radiotolerans SRS30216 was isolated from a high-level radioactive environment at the Savannah River Site (SRS) and exhibits γ-radiation resistance approaching that of Deinococcus radiodurans. The genome was sequenced by the U.S. Department of Energys Joint Genome Institute which suggested the existence of three replicons, a 4.76 Mb linear chromosome, a 0.18 Mb linear plasmid, and a 12.92 Kb circular plasmid. Southern hybridization confirmed that the chromosome is linear. The K. radiotolerans genome sequence was examined to learn about the physiology of the organism with regard to ionizing radiation resistance, the potential for bioremediation of nuclear waste, and the dimorphic life cycle. K. radiotolerans may have a unique genetic toolbox for radiation protection as it lacks many of the genes known to confer radiation resistance in D. radiodurans. Additionally, genes involved in the detoxification of reactive oxygen species and the excision repair pathway are overrepresented. K. radiotolerans appears to lack degradation pathways for pervasive soil and groundwater pollutants. However, it can respire on two organic acids found in SRS high-level nuclear waste, formate and oxalate, which promote the survival of cells during prolonged periods of starvation. The dimorphic life cycle involves the production of motile zoospores. The flagellar biosynthesis genes are located on a motility island, though its regulation could not be fully discerned. These results highlight the remarkable ability of K radiotolerans to withstand environmental extremes and suggest that in situ bioremediation of organic complexants from high level radioactive waste may be feasible.