Search

Antimicrobial resistance is an escalating threat with few new therapeutic options in the pipeline. Urinary tract infections (UTIs) are one of the most prevalent bacterial infections globally and are prone to becoming recurrent and antibiotic resistant. We discovered and characterized six novel Autographiviridae and Guernseyvirinae bacterial viruses (phage) against uropathogenic Escherichia coli (UPEC), a leading cause of UTIs. The phage genomes were between 39,471 bp - 45,233 bp, with 45.0%-51.0% GC%, and 57-84 predicted coding sequences per genome. We show that tail fiber domain structure, predicted host capsule type, and host antiphage repertoire correlate with phage host range. In vitro characterisation of phage cocktails showed synergistic improvement against a mixed UPEC strain population and when sequentially dosed. Together, these phage are a new set extending available treatments for UTI from UPEC, and phage vM_EcoM_SHAK9454 represents a promising candidate for further improvement through engineering., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacteriophage ϕX174 is a small icosahedral virus of the Microviridae with a rapid replication cycle. Previously, we found that in ϕX174 infections of Escherichia coli, the most highly upregulated host proteins are two small heat shock proteins, IbpA and IbpB, belonging to the HSP20 family, which is a universally conserved group of stress-induced molecular chaperones that prevent irreversible aggregation of proteins. Heat shock proteins were found to protect against ϕX174 lysis, but IbpA/B have not been studied. In this work, we disrupted the ibpA and ibpB genes and measured the effects on ϕX174 replication. We found that in contrast to other E. coli heat shock proteins, they are not necessary for ϕX174 replication; moreover, their absence has no discernible effect on ϕX174 fecundity. These results suggest IbpA/B upregulation is a response to ϕX174 protein expression but does not play a role in phage replication, and they are not Microviridae host factors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Viruses play important roles in modulating microbial communities and influencing global biogeochemistry. There is now growing interest in characterising their ecological roles across diverse biomes. However, little is known about viral ecology in low-nutrient, chemotrophic-based environments. In such ecosystems, virus-driven manipulation of nutrient cycles might have profound impacts across trophic levels. In particular, anchialine environments, which are low-energy underground estuaries sustained by chemotrophic processes, represent ideal model systems to study novel virus-host-environment interactions., Results: Here, we employ metagenomic sequencing to investigate the viral community in Bundera Sinkhole, an anchialine ecosystem rich in endemic species supported by microbial chemosynthesis. We find that the viruses are highly novel, with less than 2% representing described viruses, and are hugely abundant, making up as much as 12% of microbial intracellular DNA. These highly abundant viruses largely infect important prokaryotic taxa that drive key metabolic processes in the sinkhole. Further, the abundance of viral auxiliary metabolic genes (AMGs) involved in nucleotide and protein synthesis was strongly correlated with declines in environmental phosphate and sulphate concentrations. These AMGs encoded key enzymes needed to produce sulphur-containing amino acids, and phosphorus metabolic enzymes involved in purine and pyrimidine nucleotide synthesis. We hypothesise that this correlation is either due to selection of these AMGs under low phosphate and sulphate concentrations, highlighting the dynamic interactions between viruses, their hosts, and the environment; or, that these AMGs are driving increased viral nucleotide and protein synthesis via manipulation of host phosphorus and sulphur metabolism, consequently driving nutrient depletion in the surrounding water., Conclusion: This study represents the first metagenomic investigation of viruses in anchialine ecosystems, and provides new hypotheses and insights into virus-host-environment interactions in such 'dark', low-energy environments. This is particularly important since anchialine ecosystems are characterised by diverse endemic species, both in their microbial and faunal assemblages, which are primarily supported by microbial chemosynthesis. Thus, virus-host-environment interactions could have profound effects cascading through all trophic levels., (© 2024. The Author(s).)

Recent advances in genomic refactoring have been hindered by the ever-present complication of internal or cryptic transcriptional regulation. Typical approaches to these features have been to randomize or perform mass alterations to the gene sequences thought to contain the regulatory motifs; however, this approach can cause problems by altering translational speeds, introducing long distance DNA-DNA interaction effects, and inducing RNA toxicity. Previously, we developed a rational design approach named COdon Restrained Promoter SilEncing (CORPSE) which takes externally identified promoter sequences and uses position-specific scoring matrices as proxy promoter strengths to make minimal changes to promoter sequences to disable their activity. Additionally, through inverting our system we were also able to modify weak internal promoters to increase their activity. In this chapter, we augment our previous process with the biophysical model Promoter Calculator v1.0 developed by LaFleur et al. to combine promoter identification and activity prediction, with our algorithm to silently modify promoter sequences, to provide more robust promoter elimination and creation., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

With the global increase of infections caused by antibiotic-resistant bacterial strains, there is an urgent need for new methods of tackling the issue. Genomic analysis of bacterial strains can help to understand their virulence and antibiotic resistance profile. Bioinformatic skills are in great demand across the biological sciences. We designed a workshop that allows university students to learn the process of genome assembly using command-line tools within a virtual machine on a Linux operating system. We use Illumina and Nanopore short and long-read raw sequences to reveal the advantages and disadvantages of short, long, and hybrid assembly methods. The workshop teaches how to assess read and assembly quality, perform genome annotation, and analyze pathogenicity, antibiotic and phage resistance. The workshop is intended for a five-week teaching period and is concluded by a student poster presentation assessment., (© 2023 The Authors. Biochemistry and Molecular Biology Education published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

Future applications of synthetic biology will rely on deploying engineered cells outside of lab environments for long periods of time. Currently, a significant roadblock to this application is the potential for deactivating mutations in engineered genes. A recently developed method to protect engineered coding sequences from mutation is called Constraining Adaptive Mutations using Engineered Overlapping Sequences (CAMEOS). In this chapter we provide a workflow for utilizing CAMEOS to create synthetic overlaps between two genes, one essential (infA) and one non-essential (aroB), to protect the non-essential gene from mutation and loss of protein function. In this workflow we detail the methods to collect large numbers of related protein sequences, produce multiple sequence alignments (MSAs), use the MSAs to generate hidden Markov models and Markov random field models, and finally generate a library of overlapping coding sequences through CAMEOS scripts. To assist practitioners with basic coding skills to try out the CAMEOS method, we have created a virtual machine containing all the required packages already installed that can be downloaded and run locally., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Modern genome-scale methods that identify new genes, such as proteogenomics and ribosome profiling, have revealed, to the surprise of many, that overlap in genes, open reading frames and even coding sequences is widespread and functionally integrated into prokaryotic, eukaryotic and viral genomes. In parallel, the constraints that overlapping regions place on genome sequences and their evolution can be harnessed in bioengineering to build more robust synthetic strains and constructs. With a focus on overlapping protein-coding and RNA-coding genes, this Review examines their discovery, topology and biogenesis in the context of their genome biology. We highlight exciting new uses for sequence overlap to control translation, compress synthetic genetic constructs, and protect against mutation., (© 2021. Springer Nature Limited.)

Future applications of synthetic biology will require refactored genetic sequences devoid of internal regulatory elements within coding sequences. These regulatory elements include cryptic and intragenic promoters, which may constitute up to a third of the predicted Escherichia coli promoters. The promoter activity is dependent on the structural interaction of core bases with a σ factor. Rational engineering can be used to alter key promoter element nucleotides interacting with σ factors and eliminate downstream transcriptional activity. In this paper, we present codon-restrained promoter silencing (CORPSE), a system for removing intragenic promoters. CORPSE exploits the DNA-σ factor structural relationship to disrupt σ 70 promoters embedded within gene coding sequences with a minimum of synonymous codon changes. Additionally, we present an inverted CORPSE system, iCORPSE, which can create highly active promoters within a gene sequence while not perturbing the function of the modified gene.

Bacteriophage plaque size measurement is essential for phage characterisation, but manual size estimation requires a considerable amount of time and effort. In order to ease the work of phage researchers, we have developed an automated command-line application called Plaque Size Tool (PST) that can detect plaques of different morphology on the images of Petri dishes and measure plaque area and diameter. Plaque size measurements using PST showed no difference to those obtained with manual plaque size measurement in Fiji, indicating future results using PST are backwards compatible with prior measurements in the literature. PST can be applied to a range of lytic bacteriophages producing oval-shaped plaques, including bull's-eye and turbid morphology. The application can also be used for titer calculation if most of the plaques are stand-alone. As laboratory automation becomes more commonplace, standardised and flexible open-source analytical tools like PST will be important parts of biofoundry and cloud lab bacteriophage workflows., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Measuring host-bacteriophage dynamics is an important approach to understanding bacterial survival functions and responses to infection. The model Microviridae bacteriophage φX174 is endemic to the human gut and has been studied for over 70 years, but the host response to infection has never been investigated in detail. To address this gap in our understanding of this important interaction within our microbiome, we have measured host Escherichia coli C proteomic and transcriptomic response to φX174 infection. We used mass spectrometry and RNA sequencing (RNA-seq) to identify and quantify all 11 φX174 proteins and over 1,700 E. coli proteins, enabling us to comprehensively map host pathways involved in φX174 infection. Most notably, we see significant host responses centered on membrane damage and remodeling, cellular chaperone and translocon activity, and lipoprotein processing, which we speculate is due to the peptidoglycan-disruptive effects of the φX174 lysis protein E on MraY activity. We also observe the massive upregulation of small heat shock proteins IbpA/B, along with other heat shock pathway chaperones, and speculate on how the specific characteristics of holdase protein activity may be beneficial for viral infections. Together, this study enables us to begin to understand the proteomic and transcriptomic host responses of E. coli to Microviridae infections and contributes insights to the activities of this important model host-phage interaction. IMPORTANCE A major part of the healthy human gut microbiome is the Microviridae bacteriophage, exemplified by the model φX174 phage, and their E. coli hosts. Although much has been learned from studying φX174 over the last half-century, until this work, the E. coli host response to infection has never been investigated in detail. We reveal the proteomic and transcriptomic pathways differentially regulated during the φX174 infection cycle and uncover the details of a coordinated cellular response to membrane damage that results in increased lipoprotein processing and membrane trafficking, likely due to the phage antibiotic-like lysis protein. We also reveal that small heat shock proteins IbpA/B are massively upregulated during infection and that these holdase chaperones are highly conserved across the domains of life, indicating that reliance on them is likely widespread across viruses., (Copyright © 2021 Wright et al.)

Sequence overlap between two genes is common across all genomes, with viruses having high proportions of these gene overlaps. Genome modularization and refactoring is the process of disrupting natural gene overlaps to separate coding sequences to enable their individual manipulation. The biological function and fitness effects of gene overlaps are not fully understood, and their effects on gene cluster and genome-level refactoring are unknown. The bacteriophage φX174 genome has ∼26% of nucleotides involved in encoding more than one gene. In this study we use an engineered φX174 phage containing a genome with all gene overlaps removed to show that gene overlap is critical to maintaining optimal viral fecundity. Through detailed phenotypic measurements we reveal that genome modularization in φX174 causes virion replication, stability, and attachment deficiencies. Quantitation of the complete phage proteome across an infection cycle reveals 30% of proteins display abnormal expression patterns. Taken together, we have for the first time comprehensively demonstrated that gene modularization severely perturbs the coordinated functioning of a bacteriophage replication cycle. This work highlights the biological importance of gene overlap in natural genomes and that reducing gene overlap disruption should be an integral part of future genome engineering projects.

Bacteriophage ϕX174 is a model virus for studies across the fields of structural biology, genetics, gut microbiomics, and synthetic biology, but did not have a high-resolution transcriptome until this work. In this study we used next-generation sequencing to measure the RNA produced from ϕX174 while infecting its host E. coli C. We broadly confirm the past transcriptome model while revealing several interesting deviations from previous knowledge. Additionally, we measure the strength of canonical ϕX174 promoters and terminators and discover both a putative new promoter that may be activated by heat shock sigma factors, as well as rediscover a controversial Rho-dependent terminator. We also provide evidence for the first antisense transcription observed in the Microviridae, identify two promoters that may be involved in generating this transcriptional activity, and discuss possible reasons why this RNA may be produced., Competing Interests: Declaration of competing interest The authors declare no competing conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Resistance to antibiotics is an escalating global crisis, presenting a major health, social, and economic burden. An underexplored alternative to antibiotic treatment is phage therapy whereby bacteriophages are used to infect and kill pathogenic multidrug-resistant (MDR) bacteria. A primary challenge is the highly specific infectivity range of phages that can limit their ability to infect across different bacterial strains. Synthetic biology can enable the design, modification, and synthesis of phages with improved antimicrobial performance and efficacy to help realize novel strategies to study and treat bacterial infectious diseases, including those caused by MDR pathogens. In this perspective article, we discuss the potential for an innovative synthetic biology approach to enhance phage therapeutics and the role a biofoundry can play in bringing phage therapy to fruition., Competing Interests: No competing financial interests exist., (Copyright 2020, Mary Ann Liebert, Inc., publishers.)

The ability to separate, identify, and quantify proteins from complex mixtures are key foundational methods across biochemistry teaching and research. In particular, enzyme-linked immunosorbent assay (ELISA) is an important technique that is used to measure antigen concentrations in both industry and academia. There are four categories of ELISA, direct, indirect, competitive, and sandwich, each with their own applications. Sandwich ELISAs are used to determine antigen concentrations from complex mixtures of protein, such as a cell lysates, and are regularly used as medical diagnostics to diagnose illness and diseases ranging from hepatitis to celiac disease. One major problem with teaching the sandwich ELISA technique to students is the prohibitive cost due to the need to coat a 96-well plate with a capture antibody. One solution to this problem would be to significantly reduce the role of each student in the lab, but this does not adequately prepare students to perform the procedure in a research or industry lab. Instead, this laboratory exercise teaches students the procedural knowledge needed to perform a direct sandwich ELISA, but uses a simulated experience performed within a wet-lab environment. The presented scenario is the analysis of phosphorylated proteins within a synthetic signaling pathway, but because the lab uses simulated samples, it can be tailored to different topics and educational aims. The procedure is 10- to 26-fold less expensive per student to deploy than an authentic sandwich ELISA. Students in the course report that the ELISA lab significantly strengthened the connection between theory and practice. © 2019 International Union of Biochemistry and Molecular Biology, 48(1):67-73, 2020., (© 2019 International Union of Biochemistry and Molecular Biology.)

We develop a method for completing the genetics of natural living systems by which the absence of expected future discoveries can be established. We demonstrate the method using bacteriophage øX174, the first DNA genome to be sequenced. Like many well-studied natural organisms, closely related genome sequences are available-23 Bullavirinae genomes related to øX174. Using bioinformatic tools, we first identified 315 potential open reading frames (ORFs) within the genome, including the 11 established essential genes and 82 highly conserved ORFs that have no known gene products or assigned functions. Using genome-scale design and synthesis, we made a mutant genome in which all 11 essential genes are simultaneously disrupted, leaving intact only the 82 conserved but cryptic ORFs. The resulting genome is not viable. Cell-free gene expression followed by mass spectrometry revealed only a single peptide expressed from both the cryptic ORF and wild-type genomes, suggesting a potential new gene. A second synthetic genome in which 71 conserved cryptic ORFs were simultaneously disrupted is viable but with ∼50% reduced fitness relative to the wild type. However, rather than finding any new genes, repeated evolutionary adaptation revealed a single point mutation that modulates expression of gene H, a known essential gene, and fully suppresses the fitness defect. Taken together, we conclude that the annotation of currently functional ORFs for the øX174 genome is formally complete. More broadly, we show that sequencing and bioinformatics followed by synthesis-enabled reverse genomics, proteomics, and evolutionary adaptation can definitely establish the sufficiency and completeness of natural genome annotations., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Using engineered initiator tRNA for precise control of protein translation within cells has great promise within future orthogonal translation systems to decouple housekeeping protein metabolism from that of engineered genetic systems. Previously, E. coli strain C321.ΔA. exp lacking all UAG stop codons was created, freeing this "amber" stop codon for other purposes. An engineered "amber initiator" tRNA CUA fMet that activates translation at UAG codons is available, but little is known about this tRNA's orthogonality. Here, we combine for the first time the amber initiator tRNA CUA fMet in C321.ΔA. exp and measure its cellular effects. We found that the tRNA CUA fMet expression resulted in a nearly 200-fold increase in fluorescent reporter expression with a unimodal population distribution and no apparent cellular fitness defects. Proteomic analysis revealed upregulated ribosome-associated, tRNA degradation, and amino acid biosynthetic proteins, with no evidence for off-target translation initiation. In contrast to previous work, we show that UAG-initiated proteins carry N-terminal methionine, but have no evidence for glutamine. Together, our results identify beneficial features of using the amber initiator tRNA CUA fMet to control gene expression while also revealing fundamental challenges to using engineered initiator tRNAs as the basis for orthogonal translation initiation systems.

Our understanding of translation underpins our capacity to engineer living systems. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are considered as the 'start codons' for translation initiation in Escherichia coli. Translation is typically not thought to initiate from the 61 remaining codons. Here, we quantified translation initiation of green fluorescent protein and nanoluciferase in E. coli from all 64 triplet codons and across a range of DNA copy number. We detected initiation of protein synthesis above measurement background for 47 codons. Translation from non-canonical start codons ranged from 0.007 to 3% relative to translation from AUG. Translation from 17 non-AUG codons exceeded the highest reported rates of non-cognate codon recognition. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems., (Published by Oxford University Press on behalf of Nucleic Acids Research 2017.)

Wet Lab Accelerator (WLA) is a cloud-based tool that allows a scientist to conduct biology via robotic control without the need for any programming knowledge. A drag and drop interface provides a convenient and user-friendly method of generating biological protocols. Graphically developed protocols are turned into programmatic instruction lists required to conduct experiments at the cloud laboratory Transcriptic. Prior to the development of WLA, biologists were required to write in a programming language called "Autoprotocol" in order to work with Transcriptic. WLA relies on a new abstraction layer we call "Omniprotocol" to convert the graphical experimental description into lower level Autoprotocol language, which then directs robots at Transcriptic. While WLA has only been tested at Transcriptic, the conversion of graphically laid out experimental steps into Autoprotocol is generic, allowing extension of WLA into other cloud laboratories in the future. WLA hopes to democratize biology by bringing automation to general biologists.

The 5386 nucleotide bacteriophage øX174 genome has a complicated architecture that encodes 11 gene products via overlapping protein coding sequences spanning multiple reading frames. We designed a 6302 nucleotide synthetic surrogate, øX174.1, that fully separates all primary phage protein coding sequences along with cognate translation control elements. To specify øX174.1f, a decompressed genome the same length as wild type, we truncated the gene F coding sequence. We synthesized DNA encoding fragments of øX174.1f and used a combination of in vitro- and yeast-based assembly to produce yeast vectors encoding natural or designer bacteriophage genomes. We isolated clonal preparations of yeast plasmid DNA and transfected E. coli C strains. We recovered viable øX174 particles containing the øX174.1f genome from E. coli C strains that independently express full-length gene F. We expect that yeast can serve as a genomic 'drydock' within which to maintain and manipulate clonal lineages of other obligate lytic phage., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Heme and bacteriochlorophyll a (BChl) biosyntheses share the same pathway to protoporphyrin IX, which then branches as follows. Fe(2+) chelation into the macrocycle by ferrochelatase results in heme formation, and Mg(2+) addition by Mg-chelatase commits the porphyrin to BChl synthesis. It was recently discovered that a bchD (Mg-chelatase) mutant of Rhodobacter sphaeroides produces an alternative BChl in which Mg(2+) is substituted by Zn(2+). Zn-BChl has been found in only one other organism before, the acidophilic Acidiphilium rubrum. Our objectives in this work on the bchD mutant were to 1) elucidate the Zn-BChl biosynthetic pathway in this organism and 2) understand causes for the low amounts of Zn-BChl produced. The bchD mutant was found to contain a Zn-protoporphyrin IX pool, analogous to the Mg-protoporphyrin IX pool found in the wild type strain. Inhibition of ferrochelatase with N-methylprotoporphyrin IX caused Zn-protoporphyrin IX and Zn-BChl levels to decline by 80-90% in the bchD mutant, whereas in the wild type strain, Mg-protoporphyrin IX and Mg-BChl levels increased by 170-240%. Two early metabolites of the Zn-BChl pathway were isolated from the bchD mutant and identified as Zn-protoporphyrin IX monomethyl ester and divinyl-Zn-protochlorophyllide. Our data support a model in which ferrochelatase synthesizes Zn-protoporphyrin IX, and this metabolite is acted on by enzymes of the BChl pathway to produce Zn-BChl. Finally, the low amounts of Zn-BChl in the bchD mutant may be due, at least in part, to a bottleneck upstream of the step where divinyl-Zn-protochlorophyllide is converted to monovinyl-Zn-protochlorophyllide.

The α-proteobacterium Rhodobacter sphaeroides is an exemplary model organism for the creation and study of novel protein expression systems, especially membrane protein complexes that harvest light energy to yield electrical energy. Advantages of this organism include a sequenced genome, tools for genetic engineering, a well-characterized metabolism, and a large membrane surface area when grown under hypoxic or anoxic conditions. This chapter provides a framework for the utilization of R. sphaeroides as a model organism for membrane protein expression, highlighting key advantages and shortcomings. Procedures covered in this chapter include the creation of chromosomal gene deletions, disruptions, and replacements, as well as the construction of a synthetic operon using a model promoter to induce expression of modified photosynthetic reaction center proteins for structural and functional analysis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A procedure for modification of the valve stem of a 40 K French pressure cell is described. The modification should be done by a machinist and requires a metalworking lathe. After modification of the valve stem, a torlon 4203 plastic ball is used between the valve stem and valve seat to control the pressure within the cell. The torlon plastic ball is a key component needed to obtain the high pressures required for efficient disruption of microbial cells.

The cofactor composition and electron-transfer kinetics of the reaction center (RC) from a magnesium chelatase (bchD) mutant of Rhodobacter sphaeroides were characterized. In this RC, the special pair (P) and accessory (B) bacteriochlorophyll (BChl) -binding sites contain Zn-BChl rather than BChl a. Spectroscopic measurements reveal that Zn-BChl also occupies the H sites that are normally occupied by bacteriopheophytin in wild type, and at least 1 of these Zn-BChl molecules is involved in electron transfer in intact Zn-RCs with an efficiency of >95% of the wild-type RC. The absorption spectrum of this Zn-containing RC in the near-infrared region associated with P and B is shifted from 865 to 855 nm and from 802 to 794 nm respectively, compared with wild type. The bands of P and B in the visible region are centered at 600 nm, similar to those of wild type, whereas the H-cofactors have a band at 560 nm, which is a spectral signature of monomeric Zn-BChl in organic solvent. The Zn-BChl H-cofactor spectral differences compared with the P and B positions in the visible region are proposed to be due to a difference in the 5th ligand coordinating the Zn. We suggest that this coordination is a key feature of protein-cofactor interactions, which significantly contributes to the redox midpoint potential of H and the formation of the charge-separated state, and provides a unifying explanation for the properties of the primary acceptor in photosystems I (PS1) and II (PS2).

Rhodobacter capsulatus contains lhaA and pucC genes that have been implicated in light-harvesting complex 1 and 2 (LH1 and LH2) assembly. The proteins encoded by these genes, and homologues in other photosynthetic organisms, have been classified as the bacteriochlorophyll delivery (BCD) family of the major facilitator superfamily. A new BCD family phylogenetic tree reveals that several PucC, LhaA and Orf428-related sequences each form separate clusters, while plant and cyanobacterial homologues cluster more distantly. The PucC protein is encoded in the pucBACDE superoperon which also codes for LH2 alpha (PucA) and beta (PucB) proteins. PucC was previously shown to be necessary for formation of LH2. This article gives evidence indicating that PucC has a shepherding activity that keeps the homologous alpha and beta proteins of LH1 and LH2 apart, allowing LH1 to assemble properly. This shepherding function was indicated by a 62% reduction in LH1 levels in DeltaLHII strains carrying plasmids encoding pucBA along with a C-terminally truncated pucC gene. More severe reductions in LH1 were seen when the truncated pucC gene was co-expressed in the presence of C-terminal PucC::PhoA fusion proteins. It appears that interaction between truncated PucC::PhoA fusion proteins and the truncated PucC protein disrupts LH1 assembly, pointing towards a PucC dimeric or multimeric functional unit.

A Rhodobacter sphaeroides bchD (magnesium chelatase) mutant was studied to determine the properties of its photosystem in the absence of bacteriochlorophyll (BChl). Western blots of reaction center H, M, and L (RC H/M/L) proteins from mutant membranes showed levels of 12% RC H, 32% RC L, and 46% RC M relative to those of the wild type. Tricine-SDS-PAGE revealed 52% light-harvesting complex alpha chain and 14% beta chain proteins compared to those of the wild type. Pigment analysis of bchD cells showed the absence of BChl and bacteriopheophytin (BPhe), but zinc bacteriochlorophyll (Zn-BChl) was discovered. Zn-BChl binds to light-harvesting 1 (LH1) and 2 (LH2) complexes in place of BChl in bchD membranes, with a LH2:LH1 ratio resembling that of wild-type cells under BChl-limiting conditions. Furthermore, the RC from the bchD mutant contained Zn-BChl in the special pair and accessory BChl binding sites, as well as carotenoid and quinone, but BPhe was absent. Comparison of the bchD mutant RC absorption spectrum to that of Acidiphilium rubrum, which contains Zn-BChl in the RC, suggests the RC protein environment at L168 contributes to A. rubrum special pair absorption characteristics rather than solely Zn-BChl. We speculate that Zn-BChl is synthesized via the normal BChl biosynthetic pathway, but with ferrochelatase supplying zinc protoporphyrin IX for enzymatic steps following the nonfunctional magnesium chelatase. The absence of BPhe in bchD cells is likely related to Zn2+ stability in the chlorin macrocycle and consequently high resistance of Zn-BChl to pheophytinization (dechelation). Possible agents prevented from dechelating Zn-BChl include the RC itself, a hypothetical dechelatase enzyme, and spontaneous processes.