Mutagenesis of RpoE-like sigma factor genes in Bdellovibrio reveals differential control of groEL and two groES genes

BACKGROUND: Bdellovibrio bacteriovorus HD100 must regulate genes in response to a variety of environmental conditions as it enters, preys upon and leaves other bacteria, or grows axenically without prey. In addition to "housekeeping" sigma factors, its genome encodes several alternate sigma factors, including 2 Group IV-RpoE-like proteins, which may be involved in the complex regulation of its predatory lifestyle.

RESULTS: We find that one sigma factor gene, bd3314, cannot be deleted from Bdellovibrio in either predatory or prey-independent growth states, and is therefore possibly essential, likely being an alternate sigma 70. Deletion of one of two Group IV-like sigma factor genes, bd0881, affects flagellar gene regulation and results in less efficient predation, although not due to motility changes; deletion of the second, bd0743, showed that it normally represses chaperone gene expression and intriguingly we find an alternative groES gene is expressed at timepoints in the predatory cycle where intensive protein synthesis at Bdellovibrio septation, prior to prey lysis, will be occurring.

CONCLUSIONS: We have taken the first step in understanding how alternate sigma factors regulate different processes in the predatory lifecycle of Bdellovibrio and discovered that alternate chaperones regulated by one of them are expressed at different stages of the lifecycle.

In vitro and in vivo methodologies for studying the Sigma 54-dependent transcription.

Here we describe approaches and methods to assaying in vitro the major variant bacterial sigma factor, Sigma 54 (σ54), in a purified system. We include the complete transcription system, binding interactions between σ54 and its activators, as well as the self-assembly and the critical ATPase activity of the cognate activators which serve to remodel the closed promoter complexes. We also present in vivo methodologies that are used to study the impact of physiological processes, metabolic states, global signalling networks, and cellular architecture on the control of σ54-dependent gene expression.

Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype-phenotype correlations, codon bias and dominant-negative effects

The adaptor protein-2 sigma subunit (AP2sigma;2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2sigma;2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca<inf>o</inf>²⁺) homeostasis. To elucidate the role of AP2sigma;2 in Ca<inf>o</inf>²⁺ regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2sigma;2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2sigma;2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype-phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2sigma;2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2sigma;2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2sigma;2 mutations to result in a more severe FHH phenotype with genotype-phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue.

Methods for the transfer function design of multiple narrow-band bandpass sigma-delta modulators

This paper presents the design analysis of novel tunable narrow-band bandpass sigma-delta modulators, which can achieve concurrent multiple noise-shaping for multi-tone input signals. Four different design methodologies based on the noise transfer functions of comb filters, slink filters, multi-notch filters and fractional delay comb filters are applied for the design of these multiple-band sigma-delta modulators. The latter approach utilises conventional comb filters in conjunction with FIR, or allpass IIR fractional delay filters, to deliver the desired nulls for the quantisation noise transfer function. Detailed simulation results show that FIR fractional delay comb filter-based sigma-delta modulators tune accurately to most centre frequencies, but suffer from degraded resolution at frequencies close to Nyquist. However, superior accuracies are obtained from their allpass IIR fractional delay counterpart at the expense of a slight shift in noise-shaping bands at very high frequencies. The merits and drawbacks of each technique for the various sigma-delta topologies are assessed in terms of in-band signal-to-noise ratios, accuracy of tunability and coefficient complexity for ease of implementation.

The effect of clock jitter on the performance of continuous-time delta-sigma ADC for GNSS signals

Next generation Global Navigation Satellite System (GNSS) receivers will operate in multiple navigation bands. An efficient way to achieve this with lower power and cost is to employ BandPass Sampling (BPS); nevertheless, the sampling operation injects large amounts of jitter noise, which degrades the performance of the receiver. Continuous–Time (CT) Delta–Sigma (ΔΣ) modulators are capable of suppressing this noise but the impact of clock jitter at the output of the Digital– to–Analog Converter (DAC) in the feedback path of the modulator should be taken into account. This paper presents an analytical approach for describing clock jitter in GNSS receivers when a CT–ΔΣ modulator is utilized for Analog–to–Digital Conversion (ADC). The validity of the presented approach is verified through time–domain simulations using a behavioural model of the fourth–order CT–ΔΣ modulator with 1–bit NRZ DAC feedback pulse.

Narrow-band variable center frequency single-loop and multistage sigma-delta modulators for bandpass signals

Oversampled narrow-band single-loop and multistage resonator-based bandpass sigma-delta (Σ-Δ) modulators that can accommodate different passband center to sampling frequency ratios are reported. These tunable bandpass configurations are designed by analytically determining and subsequently verifying through detailed empirical simulations the required compensation hardware to deliver enhanced noise-shaping. It is demonstrated that comparatively superior in-band signal-to-noise ratios and dynamic ranges are attributed to the inclusion of appropriate digital feedforward and feedback compensators within these structures.

Application of fractional delay filters to tune the centre frequency location of single-band sigma-delta modulators

This paper presents a novel technique for the design of narrow-band sigma-delta modulators with an embedded tunable centre frequency mechanism. This method demonstrates that the use of sum filters combined with a fractional delayer provide the flexibility of tuning the noise shaping band for any desired variable centre frequency input signal.

A comparative study of complex multiple-band noise-shaping sigma-delta modulators for multi-tone input signals

This paper presents a comparative study of complex single-bit and multi-bit sigma-delta modulators that are capable of providing concurrent multiple-band noise-shaping for multi-tone narrow-band input signals. The concepts applied for the three design methodologies are based on the noise transfer functions of complex comb, complex slink and complex multi-notch filters.

Increasing the variability of centre frequency locations in multiple-band sigma-delta modulators via the use of fractional delay filters

This paper presents the design analysis of novel tunable narrow-band bandpass sigma-delta modulators, that can achieve concurrent multiple noise-shaping for multi-tone input signals. This approach utilises conventional comb filters in conjunction with FIR, or allpass IIR fractional delay filters, to deliver the desired nulls for the quantisation noise transfer function. Detailed simulation results show that FIR fractional delay comb filter based sigma-delta modulators tune accurately to most centre frequencies, but suffer from degraded resolution at frequencies close to Nyquist. However, superior accuracies are obtained from their allpass IIR fractional delay counterpart at the expense of a slight shift in noise-shaping bands at very high frequencies.